Method for selecting polynucleotides encoding antigen-specific immunoglobulin subunit

ABSTRACT

The present invention relates to a high efficiency method of expressing immunoglobulin molecules in eukaryotic cells. The invention is further drawn to a method of producing immunoglobulin heavy and light chain libraries, particularly using the trimolecular recombination method, for expression in eukaryotic cells. The invention further provides methods of selecting and screening for antigen-specific immunoglobulin molecules, and antigen-specific fragments thereof. The invention also provides kits for producing, screening and selecting antigen-specific immunoglobulin molecules. Finally, the invention provides immunoglobulin molecules, and antigen-specific fragments thereof, produced by the methods provided herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of currently pending U.S. applicationSer. No. 14/977,067, filed Dec. 21, 2015, which is a divisional of U.S.Non-provisional application Ser. No. 13/844,388, filed on Mar. 15, 2013,which claims priority benefit to U.S. Provisional Appl. No. 61/639,046,filed on Apr. 26, 2012 and U.S. Provisional Appl. No. 61/732,776, filedon Dec. 3, 2012; the content of each are hereby incorporated byreference in their entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in ASCIItext file (Name: “165547_Sequence_Listing_ascii_25.txt”; Size: 30,967bytes; and Date of Creation: Apr. 27, 2017) filed herewith isincorporated herein by reference in its entirety.

BACKGROUND Field of the Invention

The present invention relates to a high efficiency method of expressingimmunoglobulin molecules on vaccinia virus particles, e.g., EEV virions,and/or on host cells, a method of producing immunoglobulin heavy andlight chain libraries for expression in vaccinia virus particles, e.g.,EEV virions, and/or eukaryotic cells, methods of isolatingimmunoglobulins which bind specific antigens, and immunoglobulinsproduced by any of these methods. The invention also relates to fusionproteins used for expressing immunoglobulin molecules on vaccinia virusparticles, e.g., EEV virions, or on host cells.

Related Art Immunoglobulin Production

Antibodies of defined specificity are being employed in an increasingnumber of diverse therapeutic applications. A number of methods havebeen used to obtain useful antibodies for human therapeutic use. Theseinclude chimeric and humanized antibodies, and fully human antibodiesselected from libraries, e.g., phage display libraries, or fromtransgenic animals. Immunoglobulin libraries constructed inbacteriophage can derive from antibody producing cells of naïve orspecifically immunized individuals and could, in principle, include newand diverse pairings of human immunoglobulin heavy and light chains.Although this strategy does not suffer from an intrinsic repertoirelimitation, it requires that complementarity determining regions (CDRs)of the expressed immunoglobulin fragment be synthesized and foldproperly in bacterial cells. Many antigen binding regions, however, aredifficult to assemble correctly as a fusion protein in bacterial cells.In addition, the protein will not undergo normal eukaryoticpost-translational modifications. As a result, this method imposes adifferent selective filter on the antibody specificities that can beobtained. Alternatively, fully human antibodies can be isolated fromlibraries in eukaryotic systems, e.g., yeast display, retroviraldisplay, or expression in DNA viruses such as poxviruses. See, e.g.,U.S. Pat. No. 7,858,559, which is incorporated herein by reference inits entirety.

The present invention enables efficient expression of a library of fullyhuman antibodies on the surface of vaccinia virus, an envelopedmammalian virus. Similar to phage display, conditions are utilizedwherein each vaccinia virion expresses a single immunoglobulin, e.g., anantibody or scFV, on its surface.

However, in the current invention, various panning and magnetic beadbased methods have been developed to screen libraries of vaccinia-MAbvirions to select recombinant virus encoding specific antibodies. Uponinfection of mammalian cells, the antibody is not only incorporated intonewly produced virus, it is also displayed on the surface of the hostcell. This enables efficient selection strategies that combine thebenefits of selection of vaccinia-MAb virions in a cell free panningsystem, followed by cell based screening for high specificity andantibody optimization.

This is different from other technologies in the field which express asingle scFV but do not express a library. Moreover, other technologiesare designed to re-direct vaccinia infection through the scFV for genetherapy and are not used for antibody discovery. Additionally, thecurrent technology differs from the previous technology by using EEVinstead of the IMV, and also by using different fusion proteins (e.g.,A56R).

SUMMARY

In certain aspects, the disclosure is directed to fusion proteincomprising (a) a first polypeptide segment comprising a heavy chain CH1domain and (b) a second polypeptide segment comprising the transmembranedomain of a vaccinia extracellular enveloped virus (EEV)-specificmembrane protein.

In some embodiments, the fusion protein further comprising a thirdpolypeptide segment comprising an immunoglobulin heavy chain variableregion or fragment thereof. In another embodiment, the vacciniaEEV-specific membrane protein is A56R.

In certain aspects, the disclosure is directed to a polynucleotideencoding a fusion protein comprising (a) a first polypeptide segmentcomprising the human heavy chain CH1 domain and (b) a second polypeptidesegment comprising the transmembrane domain of a vaccinia extracellularenveloped virus (EEV)-specific membrane protein. In certain embodiments,the polynucleotide comprises nucleotides of SEQ ID NO: 10 which encodesamino acids 108 to 314 of A56R from Western Reserve Vaccinia virusstrain. In certain embodiments, the polynucleotide encodes amino acids215 to 421 of SEQ ID NO:11. In certain embodiments, the polynucleotidecomprises the nucleotides of SEQ ID NO: 10 which encode amino acids 215to 421 of SEQ ID NO:11.

In certain aspects, the disclosure is directed to a vector comprising apolynucleotide encoding a fusion protein comprising (a) a firstpolypeptide segment comprising the human heavy chain CH1 domain and (b)a second polypeptide segment comprising the transmembrane domain of avaccinia extracellular enveloped virus (EEV)-specific membrane protein.

In certain aspects, the disclosure is directed to a recombinant vacciniavirus comprising a polynucleotide encoding a fusion protein comprising(a) a first polypeptide segment comprising the human heavy chain CH1domain and (b) a second polypeptide segment comprising the transmembranedomain of a vaccinia extracellular enveloped virus (EEV)-specificmembrane protein. In another aspect, the disclosure is directed to ahost cell infected with the recombinant vaccinia virus.

In another aspect, the disclosure is directed to recombinant vaccinialibrary comprising a first library of polynucleotides constructed in avaccinia virus vector encoding a plurality of immunoglobulin fusionpolypeptides, wherein the vaccinia virus vector comprises (a) a firstpolynucleotide encoding a first polypeptide segment comprising a heavychain CH1 domain (b) a second polynucleotide encoding a secondpolypeptide segment comprising the transmembrane domain of a vacciniavirus EEV-specific membrane protein situated downstream of the CH1domain, and (c) a third polynucleotide encoding an immunoglobulin heavychain variable region or fragment thereof situated upstream of the CH1domain. In one embodiment, the first library further comprises a signalpeptide for facilitating expression of the fusion polypeptides on thesurface of EEV. In another embodiment, the EEV-specific membrane proteinis A56R. In another embodiment, the vaccinia EEV-specific membraneprotein is A56R. In another embodiment, the second polypeptide segmentfurther comprises the extracellular domain of the EEV-specific membraneprotein, or a portion thereof. In another embodiment, the secondpolypeptide segment further comprises the intracellular domain of theEEV-specific membrane protein, or a portion thereof. In certainembodiments, the fusion protein comprises amino acids of SEQ ID NO: 11which correspond to the polypeptide sequence amino acids 108 to 314 ofA56R from Western Reserve Vaccinia virus strain. In certain embodiments,the fusion protein comprises amino acids 215 to 421 of SEQ ID NO: 11. Incertain embodiments, the fusion protein comprises amino acids 215 to 421of SEQ ID NO: 11, which is amino acids 108 to 314 of A56R from WesternReserve Vaccinia virus strain.

In another aspect, the disclosure is directed to methods for selectingpolynucleotides which encode an antigen-specific immunoglobulin heavychain variable region or antigen-binding fragment thereof, comprising:(a) introducing the first library of any one of claims 13 to 18 encodingimmunoglobulin fusion proteins into a population of host cellspermissive for vaccinia virus infectivity; (b) introducing one or morepolynucleotides encoding an immunoglobulin light chain into thepopulation of host cells, wherein an immunoglobulin fusion protein iscapable of combining with an immunoglobulin light chain to form anantigen-binding domain of an immunoglobulin molecule; (c) permittingrelease of extracellular enveloped virus (EEV) from the host cells; (d)collecting the released EEV from the supernatant; (e) contacting thereleased EEV with an antigen; and (f) recovering the polynucleotides ofthe first library which encode the immunoglobulin fusion polypeptidesexpressed on the membrane surface of EEV and specific for the antigen.

In one embodiment, to methods for selecting polynucleotides which encodean antigen-specific immunoglobulin heavy chain variable region orantigen-binding fragment thereof further comprises: (g) introducing thepolynucleotides recovered in (f) into a second population of host cellspermissive for vaccinia virus infectivity; (h) introducing one or morepolynucleotides encoding an immunoglobulin light chain into thepopulation of host cells; (i) permitting release of extracellularenveloped virus (EEV) from the host cells; (j) collecting the releasedEEV from the supernatant; (k) contacting the released EEV with anantigen; and (l) recovering the polynucleotides of the first librarywhich encode the immunoglobulin fusion polypeptides expressed on themembrane surface of EEV and specific for the antigen.

In certain embodiments steps (g)-(l) are repeated one or more times,thereby enriching for polynucleotides of the first library which encodeimmunoglobulin heavy chain variable regions or antigen-specificfragments thereof, as part of an immunoglobulin fusion polypeptide thatspecifically binds the antigen.

In certain embodiments, the polynucleotides recovered from the firstlibrary are isolated.

In another aspect, the disclosure is directed to a method for selectingpolynucleotides which encode an antigen-specific immunoglobulin moleculeor antigen-specific fragment thereof, comprising: (a) introducing thefirst library into a population of host cells permissive for vacciniavirus infectivity; (b) introducing a second library into the populationof host cells, where in the second library comprises a plurality ofpolynucleotides encoding an immunoglobulin light chain,

Wherein the immunoglobulin fusion polypeptide is capable of combiningwith the immunoglobulin light chain to form an immunoglobulin moleculeor antigen-specific fragment thereof; (c) permitting expression of theimmunoglobulin fusion polypeptide from the host cells; (d) collectingthe immunoglobulin fusion polypeptide from the host cells; (e)contacting the collected immunoglobulin fusion polypeptide with anantigen; and (f) recovering the polynucleotides of the first librarywhich encode the immunoglobulin fusion polypeptides that are specificfor the antigen.

In one embodiment, the method for selecting polynucleotides which encodean antigen-specific immunoglobulin molecule or antigen-specific fragmentthereof further comprises: (g) introducing the polynucleotides recoveredin (f) into a second population of host cells permissive for vacciniavirus infectivity; (h) introducing into the second population of hostcells the second library of polynucleotides; (i) permitting expressionof the immunoglobulin fusion polypeptide from the host cells; (j)collecting the immunoglobulin fusion polypeptide from the host cells;(k) contacting the collected immunoglobulin fusion polypeptide with anantigen; and (l) recovering the polynucleotides of the first librarywhich encode the immunoglobulin fusion polypeptides that are specificfor the antigen.

In certain embodiments steps (g)-(l) are repeated one or more times,thereby enriching for polynucleotides of the first library which encodeimmunoglobulin heavy chain variable regions or antigen-specificfragments thereof, as part of an immunoglobulin fusion polypeptide thatspecifically binds the antigen.

In one embodiment, the a method for selecting polynucleotides whichencode an antigen-specific immunoglobulin molecule or antigen-specificfragment thereof further comprises isolating the third polynucleotidesrecovered from the first library.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1. Shows the pJEM1 plasmid elements and their respective sequences(SEQ ID NO:1).

FIG. 2. Shows an illustration of the general strategy for libraryselection using recombinant vaccinia virus.

FIG. 3A-C. Show Fluorescence Activated Cell Sorting (FACS) analysis datafor C35 staining and CD100 staining of HeLa cells infected with EEVrecombinant vaccinia virus expressing H2124-A56R+L517 (B) or2408-A56R-scFV (C) compared to wild-type (WT) infected cells (A).

FIG. 4A-B. Show ELISA binding results for EEV containing the C35specific fusion protein (labeled “A56R EEV”), a control(“L517+G7000-A56R EEV”), and C35 specific antibody in standard membranebound IgG1 format (“mbg EEV”) with C35/Anti-Vac HRP (A) and C35/Anti-Fab(B).

FIG. 5A-D. Show plaque assay plate results for C35 binding after 2 hours(A) and overnight (B), and VEGF binding after 2 hours (C) and overnight(D).

FIG. 6. Shows an illustration of the CD100 antibody selection strategy.

FIG. 7 shows an alignment of the VH sequence of CD100 clone C20 (SEQ IDNO:33) and an identical VH clone identified by the recombinant vaccinialibrary selection (SEQ ID NO:35). SEQ ID NO:34 comprises the consensussequence.

FIG. 8. Shows flow cytometry C35 and Her2 staining results for Her2.3.2and Her2.3.3 selection with light chains L48, L116, and L9021.

FIG. 9. Shows an illustration of the Her2 antibody selection strategy.

FIG. 10. Shows flow cytometry results for C35+anti-His and Her2+anti-Hisfor Her2.3.2 and Her2.3.3 selection.

FIG. 11 shows the Her2 B10 clone sequence (SEQ ID NO:20) as well as analignment of the VH sequence of Her2 clone B10 (SEQ ID NO:36) and anidentical VH clone identified by the recombinant vaccinia libraryselection (SEQ ID NO:38). SEQ ID NO:37 comprises the consensus sequence.

FIG. 12. Shows a diagram of “Fab”, “TR”, and “IgG-gamma heavy chain”constructs.

FIG. 13. Shows Fluorescence Activated Cell Sorting (FACS) analysis datafor C35 staining and Her2 staining of HeLa cells infected with EEVrecombinant vaccinia virus expressing 8000-Fab L8000.

FIG. 14. Shows Fluorescence Activated Cell Sorting (FACS) analysis datafor C35 staining and Her2 staining of HeLa cells infected with EEVrecombinant vaccinia virus expressing (A) 8000-IgG L8000 and (B) 8000-TRL8000.

FIG. 15. Shows Fluorescence Activated Cell Sorting (FACS) analysis datafor C35 staining and Her2 staining of HeLa cells infected with EEVrecombinant vaccinia virus expressing (A) H2124-IgG and (B) H2124-TRL517.

FIG. 16. Shows controls for CD100 Lib 10.3 FLOW analysis. FluorescenceActivated Cell Sorting (FACS) analysis data for Her2 staining and CD100staining of HeLa cells infected with EEV recombinant vaccinia virusexpressing (A) 2368 and (B) 8000.

FIG. 17. Shows results for Tosyl selected CD100 Lib 10.3 FLOW analysis.Fluorescence Activated Cell Sorting (FACS) analysis data for Her2staining and CD100 staining of HeLa cells infected with EEV recombinantvaccinia virus expressing (A) L223, (B) L151, and (C) L9021.

FIG. 18. Shows results for Tosyl selected CD100 Lib 10.3 FLOW analysis.Fluorescence Activated Cell Sorting (FACS) analysis data for Her2staining and CD100 staining of HeLa cells infected with EEV recombinantvaccinia virus expressing (A) L48, (B) L7110, and (C) L122.

FIG. 19. Shows results for Tosyl selected CD100 Lib 10.3 FLOW analysis.Fluorescence Activated Cell Sorting (FACS) analysis data for Her2staining and CD100 staining of HeLa cells infected with EEV recombinantvaccinia virus expressing (A) L116, (B) L214, and (C) L3-1.

FIG. 20. Shows results for ProG selected CD100 Lib 10.3 FLOW analysis.Fluorescence Activated Cell Sorting (FACS) analysis data for Her2staining and CD100 staining of HeLa cells infected with EEV recombinantvaccinia virus expressing (A) L223, (B) L151, and (C) L9021.

FIG. 21. Shows results for ProG selected CD100 Lib 10.3 FLOW analysis.Fluorescence Activated Cell Sorting (FACS) analysis data for Her2staining and CD100 staining of HeLa cells infected with EEV recombinantvaccinia virus expressing (A) L48, (B) L7110, and (C) L122.

FIG. 22. Shows results for Protein G selected CD100 Lib 10.3 FLOWanalysis. Fluorescence Activated Cell Sorting (FACS) analysis data forHer2 staining and CD100 staining of HeLa cells infected with EEVrecombinant vaccinia virus expressing (A) L116, (B) L214, and (C) L3-1.

FIG. 23. Shows controls for CD100 Lib 10.3/L3-1 FLOW analysis.Fluorescence Activated Cell Sorting (FACS) analysis data for PrecomplexHer2 staining, 2 steps CD100 staining, and Precomplex CD100 staining ofHeLa cells infected with EEV recombinant vaccinia virus expressing (A)8000 and (B) 2368.

FIG. 24. Shows results for CD100 Lib 10.3Tosyl/L3-1 FLOW analysis.Fluorescence Activated Cell Sorting (FACS) analysis data for PrecomplexHer2 staining, 2 steps CD100 staining, and Precomplex CD100 staining ofHeLa cells infected with EEV recombinant vaccinia virus expressing (A)CD100 Lib 10.3 pre-sorted Tosyl selected and (B) CD100 Lib 10.3 sortedTosyl selected.

FIG. 25. Shows results for CD100 Lib 10.3ProtG/L3-1 FLOW analysis.Fluorescence Activated Cell Sorting (FACS) analysis data for PrecomplexHer2 staining, 2 steps CD100 staining, and Precomplex CD100 staining ofHeLa cells infected with EEV recombinant vaccinia virus expressing (A)CD100 Lib 10.3 pre-sorted Protein G selected and (B) CD100 Lib 10.3sorted Protein G selected.

FIG. 26. Shows flow cytometry results showing specificity to CD100 onJurkat cells (CD100+) and BxPC3 cells for mAbs 2050, 2063, and 2110.

FIG. 27. Shows ELISA results on (A) huCD100-His coated and (B)Hemoglobin coated plates with three CD100 specific antibodies (Mab2050,MabC2063, and MabC2110) compared to positive and negative controls.

FIG. 28. Shows a schematic for identification of specific Ig-H/Ig-Lfollowing vaccinia display methods.

FIG. 29. Fluorescence Activated Cell Sorting (FACS) analysis data forC35 and Her2 staining of HeLa cells infected with EEV recombinantvaccinia virus expressing Her2 specific clones (A) D5, (B) D8, and (C)H2.

FIG. 30. Shows ELISA results for three Her2 specific antibodies(Mab8287, Mab8290, and Mab9298).

FIG. 31. Shows flow cytometry results showing specificity to Her2 onSKBR3 cells (Her2+++) for Mab8289, Mab8293, and Mab8297.

DETAILED DESCRIPTION

The present invention is broadly directed to methods of identifyingand/or producing functional, antigen-specific immunoglobulin molecules,or antigen-specific fragments (i.e., antigen-binding fragments) thereof,in a eukaryotic system displayed on the surface of extracellularenveloped vaccinia virus (EEV), as a fusion with a polypeptide segmentcomprising the transmembrane domain of an EEV-specific membrane protein.In addition, the invention is directed to methods of identifyingpolynucleotides which encode an antigen-specific immunoglobulinmolecule, or an antigen-specific fragment thereof, from complexexpression libraries of polynucleotides encoding such immunoglobulinmolecules or fragments, where the libraries are constructed and screenedin a eukaryotic system displayed on the surface of extracellularenveloped vaccinia virus (EEV), as a fusion with a polypeptide segmentcomprising the transmembrane domain of an EEV-specific membrane protein.Further embodiments include a fusion protein comprising (a) a firstpolypeptide segment comprising the human heavy chain CH1 domain (b) asecond polypeptide segment comprising the extracellular andtransmembrane domains of a vaccinia extracellular enveloped virus(EEV)-specific membrane protein. In further embodiments a fusion proteinas disclosed herein can include a binding molecule, e.g., anantigen-specific portion of an immunoglobulin or portion thereof, e.g.,a heavy chain variable region, which, when paired with a suitableimmunoglobulin light chain, binds to an antigen of interest.

One aspect of the present invention is the construction of compleximmunoglobulin libraries in a eukaryotic system displayed on the surfaceof extracellular enveloped vaccinia virus (EEV), as a fusion with apolypeptide segment comprising the transmembrane domain of anEEV-specific membrane protein.

It is to be noted that the term “a” or “an” entity, refers to one ormore of that entity; for example, “an immunoglobulin molecule,” isunderstood to represent one or more immunoglobulin molecules. As such,the terms “a” (or “an”), “one or more,” and “at least one” can be usedinterchangeably herein.

The term “eukaryote” or “eukaryotic organism” is intended to encompassall organisms in the animal, plant, and protist kingdoms, includingprotozoa, fungi, yeasts, green algae, single celled plants, multi celledplants, and all animals, both vertebrates and invertebrates. The termdoes not encompass bacteria or viruses. A “eukaryotic cell” is intendedto encompass a singular “eukaryotic cell” as well as plural “eukaryoticcells,” and comprises cells derived from a eukaryote.

The term “vertebrate” is intended to encompass a singular “vertebrate”as well as plural “vertebrates,” and comprises mammals and birds, aswell as fish, reptiles, and amphibians.

The term “mammal” is intended to encompass a singular “mammal” andplural “mammals,” and includes, but is not limited to humans; primatessuch as apes, monkeys, orangutans, and chimpanzees; canids such as dogsand wolves; felids such as cats, lions, and tigers; equids such ashorses, donkeys, and zebras, food animals such as cows, pigs, and sheep;ungulates such as deer and giraffes; rodents such as mice, rats,hamsters and guinea pigs; and bears. In certain embodiments, the mammalis a human subject.

The terms “tissue culture” or “cell culture” or “culture” or “culturing”refer to the maintenance or growth of plant or animal tissue or cells invitro under conditions that allow preservation of cell architecture,preservation of cell function, further differentiation, or all three.“Primary tissue cells” are those taken directly from tissue, i.e., apopulation of cells of the same kind performing the same function in anorganism. Treating such tissue cells with the proteolytic enzymetrypsin, for example, dissociates them into individual primary tissuecells that grow or maintain cell architecture when seeded onto cultureplates.

The term “polynucleotide” refers to any one or more nucleic acidsegments, or nucleic acid molecules, e.g., DNA or RNA fragments, presentin a nucleic acid or construct. A “polynucleotide encoding animmunoglobulin subunit polypeptide” refers to a polynucleotide whichcomprises the coding region for such a polypeptide. In addition, apolynucleotide can encode a regulatory element such as a promoter or atranscription terminator, or can encode a specific element of apolypeptide or protein, such as a secretory signal peptide or afunctional domain.

As used herein, the term “identify” refers to methods in which desiredmolecules, e.g., polynucleotides encoding immunoglobulin molecules witha desired specificity or function, are differentiated from a pluralityor library of such molecules. Identification methods include “selection”and “screening.” As used herein, “selection” methods are those in whichthe desired molecules can be directly separated from the library. Forexample, in one selection method described herein, host cells comprisingthe desired polynucleotides are directly separated from the host cellscomprising the remainder of the library by undergoing a lytic event andthereby being released from the substrate to which the remainder of thehost cells are attached. As used herein, “screening” methods are thosein which pools comprising the desired molecules are subjected to anassay in which the desired molecule can be detected. Aliquots of thepools in which the molecule is detected are then divided intosuccessively smaller pools which are likewise assayed, until a poolwhich is highly enriched from the desired molecule is achieved.

Immunoglobulins.

As used herein, an “immunoglobulin molecule” is defined as a complete,bi-molecular immunoglobulin, i.e., generally comprising four “subunitpolypeptides,” i.e., two identical heavy chains and two identical lightchains. In some instances, e.g., immunoglobulin molecules derived fromcamelid species or engineered based on camelid immunoglobulins, acomplete immunoglobulin molecule can consist of heavy chains only, withno light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448(1993). Thus, by an “immunoglobulin subunit polypeptide” is meant asingle heavy chain polypeptide or a single light chain polypeptide.Immunoglobulin molecules are also referred to as “antibodies,” and theterms are used interchangeably herein. An “isolated immunoglobulin”refers to an immunoglobulin molecule, or two or more immunoglobulinmolecules, which are substantially removed from the milieu of proteinsand other substances, and which bind a specific antigen.

The heavy chain, which determines the “class” of the immunoglobulinmolecule, is the larger of the two subunit polypeptides, and comprises avariable region and a constant region. By “heavy chain” is meant eithera full-length secreted heavy chain form, i.e., one that is released fromthe cell, or a membrane bound heavy chain form, i.e., comprising amembrane spanning domain, e.g., fusions with a polypeptide segmentcomprising the transmembrane domain of an EEV-specific membrane protein.Immunoglobulin “classes” refer to the broad groups of immunoglobulinswhich serve different functions in the host. For example, humanimmunoglobulins are divided into five classes, i.e., IgG, comprising a γheavy chain, IgM, comprising μ heavy chain, IgA, comprising an α heavychain, IgE, comprising an c heavy chain, and IgD, comprising a δ heavychain.

By “light chain” is meant the smaller immunoglobulin subunit whichassociates with the amino terminal region of a heavy chain. As with aheavy chain, a light chain comprises a variable region and a constantregion. There are two different kinds of light chains, κ and λ, and apair of these can associate with a pair of any of the various heavychains to form an immunoglobulin molecule.

Immunoglobulin subunit polypeptides typically comprise a constant regionand a variable region. In most species, the heavy chain variable region,or V_(H) domain, and the light chain variable region, or V_(L) domain,combine to form a “complementarity determining region” or CDR, theportion of an immunoglobulin molecule which specifically recognizes anantigenic epitope. A large repertoire of variable regions associatedwith heavy and light chain constant regions are produced upondifferentiation of antibody-producing cells in an animal throughrearrangements of a series of germ line DNA segments which results inthe formation of a gene which encodes a given variable region. Furthervariations of heavy and light chain variable regions take place throughsomatic mutations in differentiated cells. The structure and in vivoformation of immunoglobulin molecules is well understood by those ofordinary skill in the art of immunology. Concise reviews of thegeneration of immunoglobulin diversity can be found, e.g., in Harlow andLane, Antibodies, A Laboratory Manual Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1988) (hereinafter, “Harlow”); and Roitt, etal., Immunology Gower Medical Publishing, Ltd., London (1985)(hereinafter, “Roitt”). Harlow and Roitt are incorporated herein byreference in their entireties.

As used herein, an “antigen-specific fragment” of an immunoglobulinmolecule is any fragment or variant of an immunoglobulin molecule whichremains capable of binding an antigen. Antigen-specific fragmentsinclude, but are not limited to, Fab, Fab′ and F(ab′)₂, Fd, single-chainFvs (scFv), single-chain immunoglobulins (e.g., wherein a heavy chain,or portion thereof, and light chain, or portion thereof, are fused),disulfide-linked Fvs (sdFv), diabodies, triabodies, tetrabodies, scFvminibodies, Fab minibodies, and dimeric scFv and any other fragmentscomprising a V_(L) and a V_(H) domain in a conformation such that aspecific CDR is formed.

Antigen-specific immunoglobulin fragments can comprise the variableregion(s) alone or in combination with the entire or partial constantregion, e.g., a CH1, CH2, CH3 domain on the heavy chain, and a lightchain constant domain, e.g., a C_(κ) or C_(λ) domain, or portion thereofon the light chain. In certain aspects a fusion protein as disclosedherein comprises a heavy chain variable domain fused to a CH1 constantdomain fused to a polypeptide segment comprising the transmembranedomain of an EEV-specific membrane protein, e.g., A56R.

In certain embodiments, the present invention is drawn to methods toidentify, i.e., select or alternatively screen for, polynucleotideswhich singly or collectively encode antigen-specific immunoglobulinmolecules or antigen-specific fragments thereof. In certain embodimentsa method of selecting an immunoglobulin molecule with an antigenspecificity of interest is provided, where the immunoglobulin orantibody is displayed on the surface of an EEV, the EEV is isolated, andthe polynucleotide encoding a portion of the immunoglobulin, e.g., theVH region, is isolated.

In certain aspects, a method for selecting polynucleotides which encodean antigen-specific immunoglobulin molecule is provided, where themethod comprises: (1) introducing a first library of polynucleotidesinto a population of host cells permissive for vaccinia virusinfectivity. The library can be constructed in a vaccinia virus vector,e.g., an EEV vector, encoding a plurality of immunoglobulin fusionpolypeptides, where the vaccinia virus vector comprises (a) a firstpolynucleotide encoding a first polypeptide segment comprising the humanheavy chain CH1 domain, e.g., a CH1-gamma domain, (b) a secondpolynucleotide encoding a second polypeptide segment comprising theextracellular and transmembrane domains of a vaccinia membrane protein,e.g., a polypeptide segment comprising the transmembrane domain of anEEV-specific membrane protein, e.g., A56R, and (c) a thirdpolynucleotide encoding an immunoglobulin heavy chain variable region orfragment thereof. The method further comprises (2) introducing into thepopulation of host cells a polynucleotide encoding a light chain, e.g.,a known light chain or a second library comprising a plurality ofpolynucleotides each encoding an immunoglobulin light chain. Onceintroduced into the population of host cells, the immunoglobulin fusionpolypeptide can combine with the immunoglobulin light chain to form anantigen-binding portion of an immunoglobulin molecule, where themolecule can be expressed or “displayed” on the surface of a selectableparticle, e.g., an EEV virion produced and released by the host cellsinto the surrounding medium. The method further provides selecting EEVreleased from the host cells that bind to an antigen of interest, e.g.,by antigen-specific attachment to a plate or to beads, e.g., protein Gbeads, streptavidin beads, or tosylated beads. EEV expressing theantigen-binding domain of interest can then be recovered, and used toreinfect new host cells, thereby enriching for EEV containingpolynucleotides which encode the heavy chain of immunoglobulin bindingto the antigen of interest. The polynucleotides can then be recovered.The method can be repeated thereby enriching for polynucleotidesencoding heavy chain fusion proteins of interest.

Isolated polynucleotides encoding the immunoglobulin heavy chainpolypeptide fusion proteins binding to an antigen of interest can thenbe transferred into and expressed in host cells (either as an EEV fusionprotein or not) in which a library of polynucleotides encodingimmunoglobulin light chain variable regions fused to a polypeptidesegment comprising the transmembrane domain of an EEV-specific membraneprotein, thereby allowing identification of a polynucleotide encoding alight chain variable region which, when combined with the heavy chainvariable region identified in the first step, forms a functionalimmunoglobulin molecule, or fragment thereof, which recognizes aspecific antigen.

As used herein, a “library” is a representative genus ofpolynucleotides, i.e., a group of polynucleotides related through, forexample, their origin from a single animal species, tissue type, organ,or cell type, where the library collectively comprises at least twodifferent species within a given genus of polynucleotides. A library ofpolynucleotides can comprise at least 10, 100, 10³, 10⁴, 10⁵, 10⁶, 10⁷,10⁸, or 10⁹ different species within a given genus of polynucleotides.The genus can be related molecules, e.g., immunoglobulin variableregions, e.g., human immunoglobulin VH domains or VL domains. The VH andVL domains can represent an entire repertoire of variable domains, orcan already be antigen-specific, e.g., specific for the same antigen.More specifically, a library can encode a plurality of a immunoglobulinsubunit polypeptides, i.e., either heavy chain subunit polypeptides orlight chain subunit polypeptides. In this context, a “library” cancomprise polynucleotides of a common genus, the genus beingpolynucleotides encoding an immunoglobulin subunit polypeptide of acertain type and class e.g., a library might encode a human μ, γ1, γ-1,γ-2, γ-3, γ-4, α-1, α-2, ε, or δ heavy chain, or a human κ or λ lightchain. Although each member of any one library can encode the same heavyor light chain constant region, the library can collectively comprise atleast two, or at least 10, 100, 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, or 10⁹different variable regions i.e., a “plurality” of variable regionsassociated with the common constant region.

In other embodiments, the library can encode a plurality ofimmunoglobulin single-chain fragments which comprise a variable region,such as a light chain variable region or a heavy chain variable region,or can comprise both a light chain variable region and a heavy chainvariable region.

In one aspect, provided herein is a method to produce libraries ofpolynucleotides encoding immunoglobulin subunit polypeptides. Furtherprovided are libraries of immunoglobulin subunit polypeptidesconstructed as fusion proteins in eukaryotic expression vectors, e.g.,EEV, where the immunoglobulin subunit polypeptide is fused to apolypeptide segment comprising the transmembrane domain of anEEV-specific membrane protein, e.g., A56R.

By “recipient cell” or “host cell” or “cell” is meant a cell orpopulation of cells into which polynucleotide libraries as describedherein are introduced. Suitable host cells for libraries describedherein are eukaryotic cells permissive for vaccinia virus infection.Suitable cell lines can be vertebrate, mammalian, rodent, mouse,primate, or human cell or cell lines.

By “a population of host cells” is meant a group of cultured cells intowhich a “library” as provided herein can be introduced and expressed.Host cells for EEV libraries as described herein can be permissive forvaccinia virus infection. Host cells of the present invention can beadherent, i.e., host cells which grow attached to a solid substrate, or,alternatively, the host cells can be in suspension.

As noted above, certain methods to identify immunoglobulin moleculescomprise the introduction of a “first” library of polynucleotides(encoding, e.g., a VH-CH1-A56R fusion protein) into a population of hostcells, as well as a “second” library of polynucleotides (e.g., encodinga VL region) into the same population of host cells. The first andsecond libraries are complementary, i.e., if the “first” library encodesimmunoglobulin heavy chain variable domains, the “second” library willencode immunoglobulin light chain variable domains, thereby allowingassembly of immunoglobulin molecules, or antigen-specific fragmentsthereof, in the population of host cells, such that the immunoglobulinsare expressed, or displayed, on the surface of EEV.

Polynucleotides contained in libraries described herein can encodeimmunoglobulin subunit polypeptides through “operable association with atranscriptional control region.” One or more nucleic acid molecules in agiven polynucleotide are “operably associated” when they are placed intoa functional relationship. This relationship can be between a codingregion for a polypeptide and a regulatory sequence(s) which areconnected in such a way as to permit expression of the coding regionwhen the appropriate molecules (e.g., transcriptional activatorproteins, polymerases, etc.) are bound to the regulatory sequences(s).“Transcriptional control regions” include, but are not limited topromoters, enhancers, operators, and transcription termination signals,and are included with the polynucleotide to direct its transcription.For example, a promoter would be operably associated with a nucleic acidmolecule encoding an immunoglobulin subunit polypeptide if the promoterwas capable of effecting transcription of that nucleic acid molecule.Generally, “operably associated” means that the DNA sequences arecontiguous or closely connected in a polynucleotide. However, sometranscription control regions, e.g., enhancers, do not have to becontiguous.

By “control sequences” or “control regions” is meant DNA sequencesnecessary for the expression of an operably associated coding sequencein a particular host organism. Eukaryotic cells are known to utilizepromoters, polyadenylation signals, and/or enhancers.

A variety of transcriptional control regions are known to those skilledin the art. As will be discussed in more detail below, suitabletranscriptional control regions include promoters capable of functioningin the cytoplasm of poxvirus-infected cells.

In certain embodiments, a fusion protein as described herein cancomprise a linker, e.g., connecting the immunoglobulin variable domainto a constant domain, e.g., a CH1, C-kappa, or C-lambda domain, and/orconnecting the constant domain to a polypeptide segment comprising thetransmembrane domain of an EEV-specific membrane protein, e.g., A56R. Alinker can comprise, e.g., at least about 5, at least about 10, or atleast about 15 amino acids. Suitable linkers can be identified by aperson of ordinary skill in the art.

Where a fusion protein described herein comprises a heavy chain constantregion, e.g., a CH1 domain, any heavy chain constant region can beutilized, including, but not limited to immunoglobulin heavy chains fromvertebrates such as birds, fish, or mammals, e.g., human immunoglobulinheavy chains. For example, a human immunoglobulin heavy chains orportion thereof, e.g., a CH1 domain can be μ heavy chain or fragmentthereof, i.e., the heavy chain of an IgM immunoglobulin, a γ-1 heavychain or fragment thereof, i.e., the heavy chain of an IgG1immunoglobulin, a γ-2 heavy chain or fragment thereof, i.e., the heavychain of an IgG2 immunoglobulin, a γ-3 heavy chain or fragment thereof,i.e., the heavy chain of an IgG3 immunoglobulin, a γ-4 heavy chain orfragment thereof, i.e., the heavy chain of an IgG4 immunoglobulin, anα-1 heavy chain or fragment thereof, i.e., the heavy chain of an IgA1immunoglobulin, an α-2 heavy chain or fragment thereof, i.e., the heavychain of an IgA2 immunoglobulin, an ε heavy chain or fragment thereof,i.e., the heavy chain of an IgE immunoglobulin, or a δ heavy chain orfragment thereof, i.e., the heavy chain of an IgD immunoglobulin.

Membrane bound fusion proteins as described herein can be anchored tothe surface of a particle, e.g., a vaccinia virus particle (or virion),e.g., an EEV particle (or virion) by a transmembrane domain fused to theheavy chain polypeptide. In certain embodiments the transmembrane domainis part of a polypeptide segment comprising the transmembrane domain ofan EEV-specific membrane protein, i.e., a protein which is expressed onthe surface of an extracellular enveloped vaccinia virus, but NOT onintracellular vaccinia virus particles. In certain embodiments, theEEV-specific membrane protein, is A56R, the vaccinia HA protein. By“intracellular domain,” “cytoplasmic domain,” “cytosolic region,” orrelated terms, which are used interchangeably herein, is meant theportion of the fusion polypeptide which is inside the cell.

In those embodiments where a fusion protein or other library proteincomprises an immunoglobulin light chain or fragment thereof, anyimmunoglobulin light chain, from any animal species, can be used, e.g.,immunoglobulin light chains from vertebrates such as birds, fish, ormammals e.g., human light chains, e.g., human κ and λ light chains. Alight chain can associate with a heavy chain to produce anantigen-binding protein of an immunoglobulin molecule.

Each member of a library of polynucleotides encoding heavy chain fusionproteins as described herein can comprise (a) a first nucleic acidmolecule encoding a first polypeptide segment comprising animmunoglobulin constant region common to all members of the library,e.g., a CH1 domain, e.g., a gamma or mu CH1 domain, (b) a second nucleicacid molecule encoding a second polypeptide segment comprising theextracellular and transmembrane domains of a vaccinia extracellularenveloped virus (EEV)-specific membrane protein (e.g., A56R), where thesecond nucleic acid molecule is directly downstream and in-frame withthe first nucleic acid molecule (either directly fused or connected by alinker), and (c) an a third nucleic acid molecule encoding a thirdpolypeptide segment comprising an immunoglobulin heavy chain variableregion, where the third nucleic acid molecule is directly upstream ofand in-frame with the first nucleic acid molecule (either directly fusedor connected by a linker).

Each member of a library of polynucleotides encoding light chain fusionproteins as described herein can comprise (a) a first nucleic acidmolecule encoding a first polypeptide segment comprising animmunoglobulin constant region common to all members of the library,e.g., a C-kappa or C-lambda domain, (b) a second nucleic acid moleculeencoding a second polypeptide segment comprising the extracellular andtransmembrane domains of a vaccinia extracellular enveloped virus(EEV)-specific membrane protein (e.g., A56R), where the second nucleicacid molecule is directly downstream and in-frame with the first nucleicacid molecule (either directly fused or connected by a linker), and (c)an a third nucleic acid molecule encoding a third polypeptide segmentcomprising an immunoglobulin light chain variable region, where thethird nucleic acid molecule is directly upstream of and in-frame withthe first nucleic acid molecule (either directly fused or connected by alinker).

Libraries of immunoglobulin heavy chains or light chains that are notfused to a polypeptide segment comprising the transmembrane domain of anEEV-specific membrane protein can be used to coinfect host cells toprovide the “complementary” immunoglobulin chain to produce a functionalantigen-binding immunoglobulin fragment. Such libraries are described indetail in, e.g., U.S. Pat. No. 7,858,559.

Libraries of polynucleotides encoding heavy or light chain variableregions can contain a plurality, i.e., at least two, or at least 10,100, 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, or 10⁹ different variable regions. Asis well known by those of ordinary skill in the art, a light chainvariable region is encoded by rearranged nucleic acid molecules, eachcomprising a light chain V_(L) region, specifically a Vκ region or a Vλregion, and a light chain J region, specifically a Jκ region or a Jλregion. Similarly, a heavy chain variable region is encoded byrearranged nucleic acid molecules, each comprising a heavy chain V_(H)region, a D region and J region. These rearrangements take place at theDNA level upon cellular differentiation. Nucleic acid molecules encodingheavy and light chain variable regions can be derived, for example, byPCR from mature B cells and plasma cells which have terminallydifferentiated to express an antibody with specificity for a particularepitope. Furthermore, if antibodies to a specific antigen are desired,variable regions can be isolated from mature B cells and plasma cells ofan animal that has been immunized with that antigen, and has therebyproduced an expanded repertoire of antibody variable regions whichinteract with the antigen. Alternatively, if a more diverse library isdesired, variable regions can be isolated from precursor cells, e.g.,pre-B cells and immature B cells, which have undergone rearrangement ofthe immunoglobulin genes, but have not been exposed to antigen, eitherself or non-self. For example, variable regions can be isolated byRT-PCR from normal human bone marrow pooled from multiple donors.Alternatively, variable regions can be synthetic, for example, made inthe laboratory through generation of synthetic oligonucleotides, or canbe derived through in vitro manipulations of germ line DNA resulting inrearrangements of the immunoglobulin genes.

In addition to first and second nucleic acid molecules encodingimmunoglobulin constant regions and variable regions, respectively, eachmember of a library of polynucleotides of the present invention asdescribed above can further comprise an additional nucleic acid moleculeencoding a signal peptide directly upstream of and in frame with thenucleic acid molecule encoding the variable region.

By “signal peptide” is meant a polypeptide sequence which, for example,directs transport of nascent immunoglobulin polypeptide subunit to thesurface of the host cells. Signal peptides are also referred to in theart as “signal sequences,” “leader sequences,” “secretory signalpeptides,” or “secretory signal sequences.” Signal peptides are normallyexpressed as part of a complete or “immature” polypeptide, and arenormally situated at the N-terminus.

All cells, including host cells of the present invention, possess aconstitutive secretory pathway, where proteins, including secretedimmunoglobulin subunit polypeptides destined for export, are secretedfrom the cell. These proteins pass through the ER-Golgi processingpathway where modifications can occur. If no further signals aredetected on the protein it is directed to the cell's surface forsecretion or insertion as an integral membrane component expressed onthe surface of the host cell or virus particle, e.g., EEV virion.Membrane-bound forms of immunoglobulin subunit polypeptides initiallyfollow the same pathway as the secreted forms, passing through to the ERlumen, except that they are retained in the ER membrane by the presenceof stop-transfer signals, or “transmembrane domains.” Transmembranedomains are hydrophobic stretches of about 20 amino acid residues thatadopt an alpha-helical conformation as they transverse the membrane.Membrane embedded proteins are anchored in the phospholipid bilayer ofthe plasma membrane. As with secreted proteins, the N-terminal region oftransmembrane proteins have a signal peptide that passes through themembrane and is cleaved upon exiting into the lumen of the ER.

Newly synthesized immunoglobulin heavy chains are held in residence inthe ER by a chaperone protein called BiP (a member of the Hsp70molecular chaperone family). Pairing of the heavy chain CH1 domain withthe CL domain of its partner light chain induces dissociation of BiP,final folding and disulfide bond formation, and egress of the assembledantibody from the ER. The antibody then utilizes the normal secretionpathway of the cell, and traffics through the Golgi to the cell surface,where it is either secreted, or retained on the surface (if the antibodyhas a transmembrane domain). See Daniel et al., Molecular Cell 34:635-36(2009).

Suitable signal peptides provided herein can be either anaturally-occurring immunoglobulin signal peptides, i.e., encoded by asequence which is part of a naturally occurring heavy or light chaintranscript, or a functional derivative of that sequence that retains theability to direct the secretion of the immunoglobulin subunitpolypeptide that is operably associated with it. Alternatively, aheterologous signal peptide, or a functional derivative thereof, can beused. In certain aspects, the signal peptide can be that of the vacciniavirus A56R protein, or a functional derivative thereof.

In other aspects, members of a library of polynucleotides as describedherein can further comprise additional nucleic acid molecules encodingheterologous polypeptides. Such additional nucleic acid moleculesencoding heterologous polypeptides can be upstream of or downstream fromthe nucleic acid molecules encoding an immunoglobulin variable orconstant domain, or the EEV-specific membrane protein.

A heterologous polypeptide encoded by an additional nucleic acidmolecule can be a rescue sequence. A rescue sequence is a sequence whichcan be used to purify or isolate either the immunoglobulin or fragmentthereof or the polynucleotide encoding it. Thus, for example, peptiderescue sequences include purification sequences such as the 6-His tagfor use with Ni affinity columns and epitope tags for detection,immunoprecipitation, or FACS (fluorescence-activated cell sorting).Suitable epitope tags include myc (for use with commercially available9E10 antibody), the BSP biotinylation target sequence of the bacterialenzyme BirA, flu tags, LacZ, and GST. The additional nucleic acidmolecule can also encode a peptide linker.

The polynucleotides comprised in various libraries described herein canbe introduced into suitable host cells. Suitable host cells can becharacterized by, e.g., being capable of expressing immunoglobulinmolecules attached to their surface or by being permissive for vacciniavirus infectivity. Polynucleotides can be introduced into host cells bymethods which are well known to those of ordinary skill in the art.Where the polynucleotide is part of a virus vector, e.g., a vacciniavirus, introduction into host cells is conveniently carried out bystandard infection.

The first and second libraries of polynucleotides can be introduced intohost cells in any order, or simultaneously. For example, if both thefirst and second libraries of polynucleotides are constructed invaccinia virus vectors, whether infectious or inactivated, the vectorscan be introduced by simultaneous infection as a mixture, or can beintroduced in consecutive infections. If one library is constructed in avaccinia virus vector, and the other is constructed in a plasmid vector,introduction can be carried out by introduction of one library beforethe other.

Following introduction into the host cells of the first and secondlibraries of polynucleotides, expression of immunoglobulin molecules, orantigen-specific fragments thereof on the surface of EEV, is permittedto occur. By “permitting expression” is meant allowing the vectors whichhave been introduced into the host cells to undergo transcription andtranslation of the immunoglobulin subunit polypeptides, allowing thehost cells to transport fully assembled immunoglobulin molecules, orantigen-specific fragments thereof, to the membrane surface as a fusionwith a polypeptide segment comprising the transmembrane domain of anEEV-specific membrane protein. Typically, permitting expression requiresincubating the host cells into which the polynucleotides have beenintroduced under suitable conditions to allow expression. Thoseconditions, and the time required to allow expression will vary based onthe choice of host cell and the choice of vectors, as is well known bythose of ordinary skill in the art.

In certain embodiments, host cells and/or vaccinia virions which havebeen allowed to express immunoglobulin molecules on their surface, orsoluble immunoglobulin molecules secreted into the cell medium are thencontacted with an antigen. As used herein, an “antigen” is any moleculethat can specifically bind to an antibody, immunoglobulin molecule, orantigen-specific fragment thereof. By “specifically bind” is meant thatthe antigen binds to the CDR of the antibody. The portion of the antigenwhich specifically interacts with the CDR is an “epitope,” or an“antigenic determinant.” An antigen can comprise a single epitope, buttypically, an antigen comprises at least two epitopes, and can includeany number of epitopes, depending on the size, conformation, and type ofantigen.

Antigens are typically peptides or polypeptides, but can be any moleculeor compound. For example, an organic compound, e.g., dinitrophenol orDNP, a nucleic acid, a carbohydrate, or a mixture of any of thesecompounds either with or without a peptide or polypeptide can be asuitable antigen. The minimum size of a peptide or polypeptide epitopeis thought to be about four to five amino acids. Peptide or polypeptideepitopes can contain at least seven, at least nine, or between at leastabout 15 to about 30 amino acids. Since a CDR can recognize an antigenicpeptide or polypeptide in its tertiary form, the amino acids comprisingan epitope need not be contiguous, and in some cases, may not even be onthe same peptide chain. In the present invention, peptide or polypeptideantigens can contain a sequence of at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 15, at least20, at least 25, and between about 15 to about 30 amino acids. Incertain embodiments, peptides or polypeptides comprising, oralternatively consisting of, antigenic epitopes are at least 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 aminoacid residues in length. The antigen can be in any form and can be free,for example dissolved in a solution, or can be attached to anysubstrate. Suitable substrates are disclosed herein. In certainembodiments, an antigen can be part of an antigen-expressing vacciniavirus, e.g., EEV virion as described in more detail below.

Immunoglobulin molecules specific for any antigen can be producedaccording to the methods disclosed herein. In certain embodiments,antigens are “self” antigens, i.e., antigens derived from the samespecies as the immunoglobulin molecules produced. As an example, itmight be desired to produce human antibodies directed to human tumorantigens. Other desired “self” antigens include, but are not limited to,cytokines, receptors, ligands, glycoproteins, and hormones.

Antibodies directed to antigens on infectious agents can also beidentified and selected by the disclosed methods. Examples of suchantigens include, but are not limited to, bacterial antigens, viralantigens, parasite antigens, and fungal antigens.

In certain selection and screening schemes in which immunoglobulinmolecules are expressed on the surface of EEV, the recombinant EEVvirions produced as described are “contacted” with antigen by a methodwhich will allow an antigen, which specifically recognizes a CDR of animmunoglobulin molecule expressed on the surface of the EEV, to bind tothe CDR, thereby allowing recombinant EEV virions which specificallybind the antigen to be distinguished from those EEV virions which do notbind the antigen. Any method which allows recombinant EEV virionsexpressing an antigen-specific binding domain of an antibody to interactwith the antigen is included. For example, if the EEV virions are insuspension, and the antigen is attached to a solid substrate,recombinant EEV virions which specifically bind to the antigen will betrapped on the solid substrate, allowing those virions which do not bindthe antigen to be washed away, and the bound recombinant EEV virions tobe subsequently recovered. Methods by which to allow recombinant EEVvirions to contact antigen, are disclosed herein.

After recovery of recombinant EEV virions which specifically bindantigen, polynucleotides of the first library can be recovered fromthose EEV virions. By “recovery” is meant a crude separation of adesired component from those components which are not desired. Forexample, recombinant EEV virions which bind antigen can be “recovered”based on their attachment to antigen-coated solid substrates, e.g.,magnetic beads, which can then be separated with a magnet.

Recovery of polynucleotides can be accomplished by any standard methodknown to those of ordinary skill in the art. In certain embodiments, thepolynucleotides are recovered by harvesting infectious EEV virions whichbound antigen.

As will be readily appreciated by those of ordinary skill in the art,identification of polynucleotides encoding immunoglobulin fusionpolypeptides can require two or more rounds of selection as describedabove, and will necessarily require two or more rounds of screening asdescribed above. A single round of selection may not necessarily resultin isolation of a pure set of polynucleotides encoding the desired firstimmunoglobulin fusion polypeptides; the mixture obtained after a firstround can be enriched for the desired polynucleotides but may also becontaminated with non-target insert sequences. Accordingly, the firstselection step, as described, can, or must be repeated one or moretimes, thereby enriching for the polynucleotides encoding the desiredimmunoglobulin fusion polypeptides. In order to repeat the first step ofthis embodiment, EEV comprising those polynucleotides recovered asdescribed above, can be introduced via infection into a population ofhost cells. The second library of polynucleotides are also introducedinto these host cells, e.g, by infection with vaccinia virus capable ofexpressing the complementary immunoglobulin molecules (e.g., lightchains) encoded by the polynucleotides in the library, and expression ofimmunoglobulin molecules, or antigen-specific fragments thereof, on themembrane surface of the recombinant EEV virions, is permitted. Therecombinant EEV virions are similarly contacted with antigen, andpolynucleotides of the first library are again recovered from EEVvirions, which express an immunoglobulin molecule that specificallybinds antigen. These steps can be repeated one or more times, resultingin enrichment for polynucleotides derived from the first library whichencode an immunoglobulin fusion polypeptide which, as part of animmunoglobulin molecule, or antigen-specific fragment thereof,specifically binds the antigen and/or has a desired functionalcharacteristic.

Following suitable enrichment for the desired polynucleotides from thefirst library as described above, those polynucleotides which have beenrecovered are “isolated,” i.e., they are substantially removed fromtheir native environment and are largely separated from polynucleotidesin the library which do not encode antigen-specific immunoglobulinfusion polypeptides. For example, cloned polynucleotides contained in avector are considered isolated. It is understood that two or moredifferent immunoglobulin fusion polypeptides which, when combined with,e.g., a light chain, specifically bind the same antigen can be recoveredby the methods described herein. Accordingly, a mixture ofpolynucleotides which encode polypeptides binding to the same antigen isalso considered to be “isolated.” Further examples of isolatedpolynucleotides include those maintained in heterologous host cells, inrecombinant vaccinia, e.g., EEV virions, or purified (partially orsubstantially) DNA molecules in solution. However, a polynucleotidecontained in a clone that is a member of a mixed library and that hasnot been isolated from other clones of the library, e.g., by virtue ofencoding an antigen-specific immunoglobulin fusion polypeptide, is not“isolated” for the purposes of this invention. For example, apolynucleotide contained in a virus vector is “isolated” after it hasbeen recovered, and optionally plaque purified.

Given that an antigen can comprise two or more epitopes, and severaldifferent immunoglobulin molecules can bind to any given epitope, it iscontemplated that several suitable polynucleotides, e.g., two, three,four, five, ten, 100 or more polynucleotides, can be recovered from thefirst step of this embodiment, all of which can encode an immunoglobulinfusion polypeptide which, when combined with a suitable immunoglobulinsubunit polypeptide encoded by a preselected polynucleotide or apolynucleotide of the second library, will form an immunoglobulinmolecule, or antigen binding fragment thereof, capable of specificallybinding the antigen of interest. It is contemplated that each differentpolynucleotide recovered from the first library would be separatelyisolated.

Once one or more suitable polynucleotides from the first library areisolated, in the second step of this embodiment, one or morepolynucleotides are identified in the second library which encodeimmunoglobulin subunit polypeptides which are capable of associatingwith the immunoglobulin fusion polypeptide(s) encoded by thepolynucleotides isolated from the first library to form animmunoglobulin molecule, or antigen-binding fragment thereof, whichspecifically binds an antigen of interest.

Provided herein are vaccinia virus vectors for expression ofantigen-binding molecules, where the antigen binding molecule, e.g., animmunoglobulin heavy chain variable region and CH1, is expressed as afusion with an EEV-specific membrane protein. In certain embodiments,heavy chains can be recovered as EEV fusion proteins, and libraries oflight chains, or individual pre-selected light chains can be expressedas soluble proteins in vaccinia virus, or other vectors, e.g., plasmidvectors.

In certain aspects, inactivation of viruses expressing a solublecomplementary chain, e.g., a light chain, can be carried out with4′-aminomethyl-trioxsalen (psoralen) and then exposing the virus vectorto ultraviolet (UV) light. Psoralen and UV inactivation of viruses iswell known to those of ordinary skill in the art. See, e.g., Tsung, K.,et al., J. Virol. 70:165-171 (1996), which is incorporated herein byreference in its entirety.

The ability to assemble and express immunoglobulin molecules orantigen-specific fragments thereof in eukaryotic cells from twolibraries of polynucleotides encoding immunoglobulin subunitpolypeptides, where one subunit is expressed as a fusion with anEEV-specific membrane protein provides a significant improvement overthe methods of producing single-chain antibodies in bacterial systems,in that the two-step selection process can be the basis for selection ofimmunoglobulin molecules or antigen-specific fragments thereof with avariety of specificities.

Vaccinia EEV vectors. Poxviruses are unique among DNA viruses becausethey replicate only in the cytoplasm of the host cell, outside of thenucleus. During its replication cycle, vaccinia virus produces fourinfectious forms which differ in their outer membranes: intracellularmature virion (IMV), the intracellular enveloped virion (IEV), thecell-associated enveloped virion (CEV) and the extracellular envelopedvirion (EEV). The prevailing view is that the IMV consists of a singlelipoprotein membrane, while the CEV and EEV are both surrounded by twomembrane layers and the IEV has three envelopes. EEV is shed from theplasma membrane of the host cell and the EEV membrane is derived fromthe trans-Golgi.

After infection, the virus loses its membrane(s) and the DNA/proteincore is transported along microtubules into the cell. The proteinsencoded by early vaccinia mRNAs (“early” is defined as pre-DNAreplication) lead to uncoating of the vaccinia core and subsequent DNAreplication. This replication occurs in what are termed “viralfactories” which are located essentially on top of the ER. Within theviral factory, immature virions (IV) assemble and are processed to formIMV (Intracellular Mature Virus). IMVs contain a membrane that isderived from the ER. The majority of IMVs are released from the cell bycell lysis. Some IMVs are transported on microtubules to sites ofwrapping by membranes of the trans-Golgi network or early endosomes. Thewrapping of the IMV particles by a double membrane creates a form ofvaccinia called IEVs (Intracellular Enveloped Virus). The IEVs are thentransported to the cell surface on microtubules. The outer IEV membranefuses with the plasma membrane to expose a CEV (Cell AssociatedEnveloped Virus) at the cell surface. Actin polymerization from the hostcell can drive the CEV to infect neighboring cells, or the virus can bereleased as an EEV. See, e.g., Kim L. Roberts and Geoffrey L. Smith.Trends in Microbiology 16(10):472-479 (2008); Geoffrey L. Smith, et al.,Journal of General Virology 83:2915-2931 (2002).

At least six virus-encoded proteins have been reported as components ofthe EEV envelope. Of these, four proteins (A33R, A34R, A56R, and B5R)are glycoproteins, one (A36R) is a nonglycosylated transmembraneprotein, and one (F13L) is a palmitoylated peripheral membrane protein.See, e.g., Lorenzo et al., Journal of Virology 74(22):10535 (2000).During infection, these proteins localize to the Golgi complex, wherethey are incorporated into infectious virus that is then transported andreleased into the extracellular medium. As provided herein,immunoglobulin fusion polypeptides, e.g., variable heavy chains arebound to the EEV membrane, e.g., as a fusion protein with anEEV-specific membrane protein, e.g., A56R.

EEV fusion proteins as provided herein can be expressed in any suitablevaccinia virus. In certain embodiments, the DNA encoding an EEV fusionprotein can be inserted into a region of the vaccinia virus genome whichis non-essential for growth and replication of the vector so thatinfectious viruses are produced. Although a variety of non-essentialregions of the vaccinia virus genome have been characterized, the mostwidely used locus for insertion of foreign genes is the thymidine kinaselocus, located in the HindIII J fragment in the genome.

Libraries of polynucleotides encoding immunoglobulin fusion polypeptidesare inserted into vaccinia virus vectors, under operable associationwith a transcriptional control region which functions in the cytoplasmof a poxvirus-infected cell.

Poxvirus transcriptional control regions comprise a promoter and atranscription termination signal. Gene expression in poxviruses istemporally regulated, and promoters for early, intermediate, and lategenes possess varying structures. Certain poxvirus genes are expressedconstitutively, and promoters for these “early-late” genes bear hybridstructures. Synthetic early-late promoters have also been developed. SeeHammond J. M., et al., J. Virol. Methods 66:135-8 (1997); ChakrabartiS., et al., Biotechniques 23:1094-7 (1997). For embodiments disclosedherein, any poxvirus promoter can be used, but use of early, late, orconstitutive promoters can be desirable based on the host cell and/orselection scheme chosen. In certain embodiments, a constitutivepromoters is used. A suitable promoter for use in the methods describedherein is the early/late 7.5-kD promoter, or the early/late H5 promoter(or variants thereof).

The Tri-Molecular Recombination Method. Traditionally, poxvirus vectorssuch as vaccinia virus have not been used to identify previously unknowngenes of interest from a complex libraries because a high efficiency,high titer-producing method of constructing and screening libraries didnot exist for vaccinia. The standard methods of heterologous proteinexpression in vaccinia virus involve in vivo homologous recombinationand in vitro direct ligation. Using homologous recombination, theefficiency of recombinant virus production is in the range ofapproximately 0.1% or less. Although efficiency of recombinant virusproduction using direct ligation is higher, the resulting titer isrelatively low. Thus, the use of vaccinia virus vector has been limitedto the cloning of previously isolated DNA for the purposes of proteinexpression and vaccine development.

Tri-molecular recombination, as disclosed in Zauderer, PCT PublicationNo. WO 00/028016 and in U.S. Pat. No. 7,858,559, is a high efficiency,high titer-producing method for producing libraries in vaccinia virus.Using the tri-molecular recombination method, the present inventor hasachieved generation of recombinant viruses at efficiencies of at least90%, and titers at least at least 2 orders of magnitude higher thanthose obtained by direct ligation.

In certain embodiments, libraries of polynucleotides capable ofexpressing immunoglobulin fusion polypeptides as described herein can beconstructed in poxvirus vectors, e.g., vaccinia virus vectors, bytri-molecular recombination.

In certain embodiments, a transfer plasmid for producing libraries offusion polypeptides is provided, which comprises a polynucleotideencoding an immunoglobulin heavy chain CH1 and at least thetransmembrane portion of a vaccinia virus A56R protein through operableassociation with a vaccinia virus H5 promoter. An exemplary vector ispromoter is pJEM1, which comprises the sequence:

AAAAAATGAAAATAAATACAAAGGTTCTTGAGGGTTGTGTTAAATTGAAAGCGAGAAATAATCATAAAT

GAAGAATACTCCACAGAGTTGATTGTAAATACAGATAGTGAATCGACTATAGACATAATACTATCTGGATCTACACATTCACCGGAAACTAGTTCTAAGAAACCTGATTATATAGATAATTCTAATTGCTCGTCGGTATTCGAAATCGCGACTCCGGAACCAATTACTGATAATGTAGAAGATCATACAGACACCGTCACATACACTAGTGATAGCATTAATACAGTAAGTGCATCATCTGGAGAATCCACAACAGACGAGACTCCGGAACCAATTACTGATAAAGAAGATCATACAGTTACAGACACTGTCTCATACACTACAGTAAGTACATCATCTGGAATTGTCACTACTAAATCAACCACCGATGATGCGGATCTTTATGATACGTACAATGATAATGATACAGTACCACCAACTACTGTAGGCGGTAGTACAACCTCTATTAGCAATTATAAAACCAAGGACTTTGTAGAAATATTTGGTATTACCGCATTAATTATATTGTCGGCCGTGGCAATTTTCTGTATTACATATTATATATATAATAAACGTTCACGTAAATACAAAACAGAGAACAAAGTCTAG Double underline - H5 promoterSingle underline - Leader peptide

No underline - Vaccinia A56R Bold italics - BssHII and BstEII variablegene cloning sitedesignated herein as SEQ ID NO:1. Various different PCR-amplified heavychain variable regions can be inserted in-frame into unique BssHII andBstEII sites, which are indicated above in bold italics.

Plasmid pJEM1 is a derivative of p7.5/tk described in U.S. Pat. No.7,858,559. pJEM1 retains the flanking regions of homology to thevaccinia genome which enables recombination as is described in U.S. Pat.No. 7,858,559. However, in place of the expression cassette in p7.5/tk(promoter and expressed sequences), pJEM1 contains the followingelements:

-   -   Vaccinia Virus H5 Promoter    -   Leader Peptide    -   5′ BssHII Cloning site for cloning variable heavy chains    -   Heavy Variable region    -   3′ BstEII Cloning site for cloning variable heavy chains    -   IgG CH1 domain        -   vaccinia A56R

These elements are listed in FIG. 1 and SEQ ID NO: 1. This cassette canbe created synthetically.

In another embodiment, the transfer plasmid of the present inventionwhich comprises a polynucleotide encoding an immunoglobulin kappa lightchain polypeptide through operable association with a vaccinia virusp7.5 promoter is pVKE, which comprises the sequence:

GGCCAAAAATTGAAAAACTAGATCTATTTATTGCACGCGGCCGCCCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGC

T TGA

ATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGGTCGACdesignated herein as SEQ ID NO:2. PCR-amplified kappa light chainvariable regions can be inserted in-frame into unique ApaLI), and XhoIsites, which are indicated above in bold.

Furthermore, pVKE can be used in those embodiments where it is desiredto have polynucleotides of the second library in a plasmid vector duringthe selection of polynucleotides of the first library as describedabove.

In another embodiment, the transfer plasmid of the present inventionwhich comprises a polynucleotide encoding an immunoglobulin lambda lightchain polypeptide through operable association with a vaccinia virusp7.5 promoter is pVLE, which comprises the sequence:

GGCCAAAAATTGAAAAACTAGATCTATTTATTGCACGCGGCCGCCCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGC GTGCAC TTGACTCGAG AAGCTTACCGTCCTACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGG TCGACdesignated herein as SEQ ID NO:3. PCR-amplified lambda light chainvariable regions can be inserted in-frame into unique ApaLI and HindIIIsites, which are indicated above in bold.

Furthermore, pVLE can be used in those embodiments where it is desiredto have polynucleotides of the second library in a plasmid vector duringthe selection of polynucleotides of the first library as describedabove.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2nd Ed., Sambrook et al., ed., Cold SpringHarbor Laboratory Press: (1989); Molecular Cloning: A Laboratory Manual,Sambrook et al., ed., Cold Springs Harbor Laboratory, New York (1992),DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); OligonucleotideSynthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195;Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984);Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984);Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987);Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A PracticalGuide To Molecular Cloning (1984); the treatise, Methods In Enzymology(Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells(J. H. Miller and M. P. Calos eds., 1987, Cold Spring HarborLaboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.),Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker,eds., Academic Press, London, 1987); Handbook Of ExperimentalImmunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986);Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986); and in Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1989).

General principles of antibody engineering are set forth in AntibodyEngineering, 2nd edition, C. A. K. Borrebaeck, Ed., Oxford Univ. Press(1995). General principles of protein engineering are set forth inProtein Engineering, A Practical Approach, Rickwood, D., et al., Eds.,IRL Press at Oxford Univ. Press, Oxford, Eng. (1995). General principlesof antibodies and antibody-hapten binding are set forth in: Nisonoff,A., Molecular Immunology, 2nd ed., Sinauer Associates, Sunderland, Mass.(1984); and Steward, M. W., Antibodies, Their Structure and Function,Chapman and Hall, New York, N.Y. (1984). Additionally, standard methodsin immunology known in the art and not specifically described aregenerally followed as in Current Protocols in Immunology, John Wiley &Sons, New York; Stites et al. (eds), Basic and Clinical-Immunology (8thed.), Appleton & Lange, Norwalk, Conn. (1994) and Mishell and Shiigi(eds), Selected Methods in Cellular Immunology, W. H. Freeman and Co.,New York (1980).

Standard reference works setting forth general principles of immunologyinclude Current Protocols in Immunology, John Wiley & Sons, New York;Klein, J., Immunology: The Science of Self-Nonself Discrimination, JohnWiley & Sons, New York (1982); Kennett, R., et al., eds., MonoclonalAntibodies, Hybridoma: A New Dimension in Biological Analyses, PlenumPress, New York (1980); Campbell, A., “Monoclonal Antibody Technology”in Burden, R., et al., eds., Laboratory Techniques in Biochemistry andMolecular Biology, Vol. 13, Elsevier, Amsterdam (1984).

EXAMPLES Example 1: Preparation of CH1-A56R Fusion Protein

Heavy Chain fusion proteins were constructed to facilitate selection ofspecific immunoglobulin segments expressed on the cell surface ofrecombinant vaccinia virus.

An expression vector encoding a fusion protein including the human heavychain CH1 domain of C gamma fused to the extracellular and transmembranedomains of A56R from Western Reserve Vaccinia virus, designated hereinas CH1-A56R, as well as a C35-specific VH (H2124) was constructed by thefollowing method.

pJEM1. An expression vector comprising a polynucleotide sequenceencoding the human gamma immunoglobulin constant region (CH1), afragment of vaccinia A56R, and a cassette for insertion of a human heavychain variable region (e.g., H2124), designated herein as “pJEM1” wasconstructed. In short, p7.5/tk, produced as described in PCT PublicationNo. WO 00/028016, incorporated herein by reference in its entirety, wasconverted into pJEM1 by the following method.

IgG CH1. A cDNA coding for the human IgG heavy chain was isolated frombone marrow RNA using SMART™ RACE cDNA Amplification Kit available fromClontech, Palo Alto, Calif. The PCR was carried out using the 5′ primerhuCγ1-5B: 5′ ATTAGGATCC GGTCACCGTC TCCTCAGCC 3′ (SEQ ID NO:4), and 3′primer huCγ1-3S: 5′ ATTAGTCGAC TCATTTACCC GGAGACAGGG AGAG 3′ (SEQ IDNO:5). The PCR product comprised the following elements:BamHI-BstEII-(nucleotides encoding amino acids 111-113 ofVH)-(nucleotides encoding amino acids 114-478 of Cγ1)-TGA-SalI. Thisproduct was subcloned into pBluescriptII/KS at BamHI and SalI sites, anda second BstEII site corresponding to amino acids 191 and 192 within theCH1 domain of Cγ1 was removed by site-directed mutagenesis withoutchange to the amino acid sequence. Plasmid pBluescriptII/KS was digestedwith BstEII and SalI and the smaller DNA fragment of about 1 Kb was gelpurified. This smaller fragment was then used as a template in a PCRreaction using forward primer CH1(F)-5′-CAAGGGACCC TGGTCACCGT CTCCTCAGCCTCC-3′ (SEQ ID NO:6) (BstEII restriction site in italics and underlined)and reverse primer CH1(R) 5′-AACTTTCTTG TCCACCTTGG TGTTG-3′ (SEQ IDNO:7). The resulting PCR product of about 320 base pairs was gelpurified.

Full Length IgG. A cDNA coding for the human IgG heavy chain wasisolated from bone marrow RNA using SMART™ RACE cDNA Amplification Kitavailable from Clontech, Palo Alto, Calif. The PCR was carried out usingthe 5′ primer huCγ1-5B: (SEQ ID NO:4), and 3′ primer huCγ1-3S: (SEQ IDNO:5). The PCR product comprised the following elements:BamHI-BstEII-(nucleotides encoding amino acids 111-113 ofVH)-(nucleotides encoding amino acids 114-478 of Cγ1)-TGA-SalI. Thisproduct was subcloned into pBluescriptII/KS at BamHI and SalI sites, anda second BstEII site corresponding to amino acids 191 and 192 within theCH1 domain of Cγ1 was removed by site-directed mutagenesis withoutchange to the amino acid sequence. Plasmid pBluescriptII/KS was digestedwith BstEII and SalI and the 993 base pair DNA fragment corresponding tofull length IgG1 was gel purified.

A56R (longer form). A DNA fragment encoding amino acids 108 to 314 ofthe A56R hemagglutinin protein from vaccinia virus (Western Reserve),which comprises the stalk, transmembrane, and intracellular domains(GenBank accession No. YP_233063) was amplified from isolated WesternReserve Vaccinia Virus DNA with forward primer A56R(F) 5′-CAACACCAAGGTGGACAAGA AAGTTACATC AACTACAAAT GACACTGATA G-3′ (SEQ ID NO:8) andreverse primer A56R(R) 5′-TATAGTCGAC CTAGACTTTG TTCTCTGTTT TGTATTTACG-3′(SEQ ID NO:9) (SalI restriction site in italics and underlined). Theresulting PCR product of about 660 base pairs was gel purified.

A56R (shorter form). A DNA fragment encoding amino acids 240 to 314 ofthe A56R hemagglutinin protein from vaccinia virus (Western Reserve),which comprises the stalk, transmembrane, and intracellular domains(GenBank accession No. YP_233063) was amplified from isolated WesternReserve Vaccinia Virus DNA with forward primer A56R(F2): 5′-CAACACCAAGGTGGACAAGA AAGTTACCAC CGATGATGCG GATCTTTATG A-3′ (SEQ ID NO:21) andreverse primer A56R(R): (SEQ ID NO:9) The resulting PCR product of about263 base pairs was gel purified.

The Fab construct (IgG CH1 with A56R longer form). The 320 and 660-basepair fragments were then combined by SOE PCR using forward primer CH1(F)(SEQ ID NO:6) and reverse primer CH1 (R2): 5′-ACAAAAGTAT TGGTAATCGTGTCATAACT TTCTTGTCCA CCTTGGTGTT G-3′ (SEQ ID NO:22) for the 5′ productand A56R (F) (SEQ ID NO:8) in combination with A56R(R) (SEQ ID NO:9) forthe 3′ product. These two products were then combined to produce afusion fragment of about 980 base pairs. This fragment was digested withBstEII and SalI, and the resulting 934-base pair fragment was gelpurified.

The TR construct (Full Length IgG1 with A56R shorter form). The 993 and263-base pair fragments were combined by SOE PCR using forward primerCH1(F): (SEQ ID NO:6) and reverse primer A56R(R2): 5′-TCATAAAGATCCGCATCATC GGTGGTTTTA CCCGGAGACA GGGAGAGGCT C-3′ (SEQ ID NO:23) for the5′ product and A56R(F3): 5′-GAGCCTCTCC CTGTCTCCGG GTAAAACCAC CGATGATGCGGATCTTTATG A-3′ (SEQ ID NO:24) in combination with A56R(R): (SEQ IDNO:9) for the 3′ product. These two products were then combined toproduce a fusion fragment of about 1256 base pairs. This fragment wasdigested with BstEII and SalI, and the resulting 1235-base pair fragmentwas gel purified.

The FL construct (Full Length IgG1 with A56R longer form). The 993 and660-base pair fragments were combined by SOE PCR using forward primerCH1(F): (SEQ ID NO:6) and reverse primer A56R(R3): 5′-TATCAGTGTCATTTGTAGTT GATGTTTTAC CCGGAGACA GGGAGAGGCT C-3′ (SEQ ID NO:25) for the5′ product and A56R (F4): 5′-GAGCCTCTCC CTGTCTCCGG GTAAAACATC AACTACAAATGACACTGATA-3′ (SEQ ID NO:26) in combination with A56R(R) (SEQ ID NO:9)for the 3′ product. These two products were then combined to produce afusion fragment of about 1653-base pairs. This fragment was digestedwith BstEII and SalI, and the resulting 1632-base pair fragment was gelpurified.

Plasmid p7.5/tk was also digested with BstEII and SalI, and the largerresulting fragment of about 5.7 Kb was gel purified. These twoBstEII/SalI fragments were then ligated to produce the pJEM1 plasmid.

pJEM1 retains the flanking regions of homology to the vaccinia genomewhich enables recombination. However, in place of the expressioncassette in p7.5/tk (promoter and expressed sequences), pJEM1 containsthe following elements: Vaccinia Virus H5 promoter; Leader peptide; 5′BssHII Cloning site for cloning variable heavy chains; Heavy Variableregion; 3′ BstEII Cloning site for cloning variable heavy chains; IgGCH1 domain; and Vaccinia A56R, the sequence for these elements of pJEM1are shown in FIG. 1 and SEQ ID NO:1.

The heavy chain variable region (H2124), specific for C35, was insertedinto the BssHII and BstEII sites of pJEM1 producing a VH(H2124)-CH1-A56R fusion construct. The nucleotide and amino acidsequences for the VH (H2124)-CH1-A56R fusion construct prepared in pJEM1are shown below, respectively.

Polynucleotide Sequence Encoding VH (H2124)-CH1-A56R Fab Product FusionProtein (SEQ ID NO:10):

CAGGTGCAGCTGCAGCAGTGGGGCGCAGGACTGCTGAAGCCTAGCGAGACCCTGTCCCTCACCTGCGCTGTCTATGGCTACTCCATCACCAGCGGCTATTTCTGGAACTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGTACATCAGCTACGACGGCAGCAGCAACTCCAACCCATCTCTCAAAAATAGGGTCACAATCAGCAGAGACACCTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCCGACACCGCTGTGTATTACTGTGCCAGAGGAACTACCGGGTTTGCTTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTCGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTACATCAACTACAAATGACACTGATAAAGTAGATTATGAAGAATACTCCACAGAGTTGATTGTAAATACAGATAGTGAATCGACTATAGACATAATACTATCTGGATCTACACATTCACCGGAAACTAGTTCTAAGAAACCTGATTATATAGATAATTCTAATTGCTCGTCGGTATTCGAAATCGCGACTCCGGAACCAATTACTGATAATGTAGAAGATCATACAGACACCGTCACATACACTAGTGATAGCATTAATACAGTAAGTGCATCATCTGGAGAATCCACAACAGACGAGACTCCGGAACCAATTACTGATAAAGAAGATCATACAGTTACAGACACTGTCTCATACACTACAGTAAGTACATCATCTGGAATTGTCACTACTAAATCAACCACCGATGATGCGGATCTTTATGATACGTACAATGATAATGATACAGTACCACCAACTACTGTAGGCGGTAGTACAACCTCTATTAGCAATTATAAAACCAAGGACTTTGTAGAAATATTTGGTATTACCGCATTAATTATATTGTCGGCCGTGGCAATTTTCTGTATTACATATTATATATATAATAAACGTTCACGTAAATACAAAAC AGAGAACAAAGTCTAG

The nucleotide sequence encoding the VH (H2124) and CH1 domain isunderlined, and the nucleotide sequence encoding the A56R domain isdouble underlined.

Amino Acid Sequence of VH (H2124)-CH1-A56R Fab Product Fusion Protein(SEQ ID NO:11):

QVQLQQWGAGLLKPSETLSLTCAVYGYSITSGYFWNWIRQPPGKGLEWIGYISYDGSSNSNPSLKNRVTISRDTSKNQFSLKLSSVTAADTAVYYCARGTTGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVTSTTNDTDKVDYEEYSTELIVNTDSESTIDIILSGSTHSPETSSKKPDYIDNSNCSSVFEIATPEPITDNVEDHTDTVTYTSDSINTVSASSGESTTDETPEPITDKEDHTVTDTVSYTTVSTSSGIVTTKSTTDDADLYDTYNDNDTVPPTTVGGSTTSISNYKTKDFVEIFGITALIILSAVAIFCITYYIYNKRSRKYKTENKV.

The amino acid sequence for the VH (H2124) and CH1 domain is underlined,and the amino acid sequence for the A56R domain is double underlined.

Polynucleotide Sequence Encoding VH (H2124)-IgG-A56R TR Construct FusionProtein (SEQ ID NO:27):

CAGGTGCAGCTGCAGCAGTGGGGCGCAGGACTGCTGAAGCCTAGCGAGACCCTGTCCCTCACCTGCGCTGTCTATGGCTACTCCATCACCAGCGGCTATTTCTGGAACTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGTACATCAGCTACGACGGCAGCAGCAACTCCAACCCATCTCTCAAAAATAGGGTCACAATCAGCAGAGACACCTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCCGACACCGCTGTGTATTACTGTGCCAGAGGAACTACCGGGTTTGCTTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTCGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAACCACCGATGATGCGGATCTTTATGATACGTACAATGATAATGATACAGTACCACCAACTACTGTAGGCGGTAGTACAACCTCTATTAGCAATTATAAAACCAAGGACTTTGTAGAAATATTTGGTATTACCGCATTAATTATATTGTCGGCCGTGGCAATTTTCTGTATTACATATTATATATATAATAAACGTTCACGTAAATACAAAAC AGAGAACAAAGTCTAG

The nucleotide sequence encoding the VH (H2124) and full length Igdomain is underlined, and the nucleotide sequence encoding the shorterform A56R domain is double underlined.

Amino Acid Sequence of VH (H2124)-IgG-A56R TR Construct Fusion Protein(SEQ ID NO:28):

QVQLQQWGAGLLKPSETLSLTCAVYGYSITSGYFWNWIRQPPGKGLEWIGYISYDGSSNSNPSLKNRVTISRDTSKNQFSLKLSSVTAADTAVYYCARGTTGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKTTDDADLYDTYNDNDTVPPTTVGGSTTSISNYKTKDFVEIFGITALIILSAVAIFCITYYIYNKRSRKYKTENKV.

The amino acid sequence for the VH (H2124) and full length Ig domain isunderlined, and the amino acid sequence for the shorter form A56R domainis double underlined.

Polynucleotide Sequence Encoding VH (H2124)-IgG-A56R FL Construct FusionProtein (SEQ ID NO:29):

CAGGTGCAGCTGCAGCAGTGGGGCGCAGGACTGCTGAAGCCTAGCGAGACCCTGTCCCTCACCTGCGCTGTCTATGGCTACTCCATCACCAGCGGCTATTTCTGGAACTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGTACATCAGCTACGACGGCAGCAGCAACTCCAACCCATCTCTCAAAAATAGGGTCACAATCAGCAGAGACACCTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCCGACACCGCTGTGTATTACTGTGCCAGAGGAACTACCGGGTTTGCTTACTGGGGCCAAGGGACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTCGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAACATCAACTACAAATGACACTGATAAAGTAGATTATGAAGAATACTCCACAGAGTTGATTGTAAATACAGATAGTGAATCGACTATAGACATAATACTATCTGGATCTACACATTCACCGGAAACTAGTTCTAAGAAACCTGATTATATAGATAATTCTAATTGCTCGTCGGTATTCGAAATCGCGACTCCGGAACCAATTACTGATAATGTAGAAGATCATACAGACACCGTCACATACACTAGTGATAGCATTAATACAGTAAGTGCATCATCTGGAGAATCCACAACAGACGAGACTCCGGAACCAATTACTGATAAAGAAGATCATACAGTTACAGACACTGTCTCATACACTACAGTAAGTACATCATCTGGAATTGTCACTACTAAATCAACCACCGATGATGCGGATCTTTATGATACGTACAATGATAATGATACAGTACCACCAACTACTGTAGGCGGTAGTACAACCTCTATTAGCAATTATAAAACCAAGGACTTTGTAGAAATATTTGGTATTACCGCATTAATTATATTGTCGGCCGTGGCAATTTTCTGTATTACATATTATATATATAATAAACGTTCACGTAAATACAAAACAGAG AACAAAGTCTAG.

The nucleotide sequence encoding the VH (H2124) and full length Igdomain is underlined, and the nucleotide sequence encoding the longerform A56R domain is double underlined.

Amino Acid Sequence of VH (H2124)-IgG-A56R FL Construct Fusion Protein(SEQ ID NO:30):

QVQLQQWGAGLLKPSETLSLTCAVYGYSITSGYFWNWIRQPPGKGLEWIGYISYDGSSNSNPSLKNRVTISRDTSKNQFSLKLSSVTAADTAVYYCARGTTGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKTSTTNDTDKVDYEEYSTELIVNTDSESTIDIILSGSTHSPETSSKKPDYIDNSNCSSVFEIATPEPITDNVEDHTDTVTYTSDSINTVSASSGESTTDETPEPITDKEDHTVTDTVSYTTVSTSSGIVTTKSTTDDADLYDTYNDNDTVPPTTVGGSTTSISNYKTKDFVEIFGITALIILSAVAIFCITYYIYNKRSRKYKTE NKV

The amino acid sequence for the VH (H2124) and full length Ig domain isunderlined, and the amino acid sequence for the longer form A56R domainis double underlined.

Example 2: Expression of A56R Fusion Protein on Surface of Hela Cells

HeLa cells were infected or co-infected with recombinant EEV vacciniavirus expressing immunoglobulin fusion constructs, Variable Heavy(H2124) CH1-A56R (described in the Example above) and Ig-K (“A56R H+L”)or scFv-A56R (“A56R scFv”). An illustration of the general strategy forinfection of cells with recombinant EEV vaccinia virus and thesubsequent library selection steps is shown in FIG. 2. In the currentexample, instead of using libraries, the HeLa cells were co-infectedwith recombinant vaccinia virus expressing the VH (H2124) CH1-A56RFusion and recombinant vaccinia virus expressing the Ig-K (A56R H+L) orinfected with recombinant vaccinia virus expressing scFv-A56R.Fluorescence Activated Cell Sorting (FACS) analysis for C35 staining andCD100 staining of cells infected with EEV recombinant vaccinia virus wasperformed. Briefly, 1 μg/ml CD100-His or 1 μg/ml C35-His were added tothe samples and incubated for 30 minutes on ice. The cells were thenwashed and anti-his APC was added and the samples were incubated for 30minutes on ice, and then the samples were washed, fixed and analyzed.The FACS data is shown in FIG. 3A-C. These results show that the A56Rfusion proteins, were expressed on the cell surface.

The clones in EEV format were also tested by ELISA. Purified C35 proteinwas coated on a 96-well ELISA plate (Nunc-MaxiSorp 96 well flat bottomimmune plate Cat #439454) at 1 μg/mL in carbonate buffer. The plate waswashed and then blocked with 1×PBS, 10% FBS. Psoralen-inactivated EEVwas then added to the plate, diluted in 1×PBS/10% FBS/0.05% Tween-20,into designated wells and allowed to bind. Viral particles were detectedusing Rabbit anti-Vaccinia-HRP conjugated antibody (AbCam Catalog#28250), the antibody was diluted 1:2000 in 1×PBS/10% FBS/0.05%Tween-20. TMB substrate (to detect horseradish peroxidase (HRP)activity) was then added to the plate; color was allowed to develop andthen the reaction was terminated with an equal volume of 2N H₂SO₄. Theplate was read on an ELISA plate reader and the results are shown inFIG. 4A. A second ELISA was used to confirm binding by detecting FAb,using the same conditions as above, except the secondary antibody wasGoat anti-human IgG F(ab′)₂-HRP conjugated (Jackson ImmunoResearchcatalog #109-036-097) used at a 1:10,000 dilution of the stock antibody,with 1×PBS/10% FBS/0.05% Tween-20 as dilution buffer. Results are shownin FIG. 4B. Wells that were positive on both plates showed that theantibody construct was expressed in the presence of the vaccinia viralvirion. As shown in FIG. 3A-B, EEV containing the C35 specific fusionprotein (labeled “A56R EEV”) bound to C35, while a control(“L517+G7000-A56R EEV”), non-C35 binding EEV, did not bind, and C35specific antibody in standard membrane bound IgG1 format (“mbg EEV”)also did not bind. The data demonstrated antigen specific binding of EEVwhen the antibody was expressed with A56R.

Example 3: Plate Based and Solution Based Fusion Protein Selection

Recombinant vaccinia virus expressing A56R fusions with known C35 andVEGF binding molecules were tested for binding to target molecules usinga panning based assay. Recombinant EEV expressing immunoglobulinmolecules known to be specific for C35 (scFv2408-A56R, H2124-L517-A56Rdouble gene, and L517+H2124-A56R co-infection) or VEGF(L7000+H7000-A56R) were produced in BSC1 cells (for about 24 hours).H2124-L517-A56R double gene produced the same antibody asL517+H2124-A56R, except the Ig-H and Ig-K genes were encoded by the samevirus from the double gene and the Ig-H and Ig-K genes were encoded byseparate viruses used for the H2124-A56R co-infection.

The clones in EEV format were tested by plaque assay. Sterile 96-wellELISA plates were coated with 1 μg/ml C35 or 1 μg/ml VEGF). EEVcontaining supernatant “Neat” (undiluted) was diluted by serial dilutiongenerating 1:10 to 1:10⁶ dilutions. 100 μl of the various virusconstructs was added to designated wells and binding was allowed toproceed for 2 hours or overnight at room temperature. The cells werewashed 10 times with PBS to remove unbound EEV and then approximately25,000 BSC1 cells were added to each well, and the plates were incubatedat 37° C. overnight. Plaque formation was detected by staining the wellswith crystal violet. FIGS. 5A-D show the plaque assay plate results forC35 binding after 2 hours and overnight, and VEGF binding after 2 hoursand overnight, respectively. These results showed that the A56R fusionproteins were expressed at the surface of the vaccinia virions.Furthermore, the results showed that known binding segments producedusing A56R fusions expressed on EEV were able to bind to their specifictargets.

Next, bead-based selection was performed using Streptavidin beads,Protein G beads or tosyl activated beads.

Streptavidin (SAV) Bead Selection.

Magnetic bead-based selection was tested using recombinant EEVexpressing MAb 2408 (H2124-A56R+L517 (C35-specific)) or MAb 7000(H7000-A56R+L7000 (VEGF-specific)). Hela cells were infected with thevarious virus constructs in two T175 flasks for 2 days, supernatant wascollected, and cells were pelleted. The EEV was pelleted by spinning for1 hour in a SA-600 rotor at 15,000 RPM. The EEV pellet was resuspendedin 1 ml DMEM supplemented with 10% FBS. For each recombinant virus, 500μl supernatant (˜10{circumflex over ( )}7 pfu) was used. Next, 500 μlDMEM containing 1 μg biotin-C35 was added to each sample (resulting in asolution of 1 ml volume at 1 μg/ml concentration). The solution wasincubated in a cold room on a rotator for 2 hours. 200 μl M280Streptavidin (SAV) magnetic beads were added to the EEV/C35 solution(the SAV bead concentration was high enough to bind all of thebiotin-C35 so no washing step was required). The solution was rotated atroom temp for 20 minutes to allow the beads to bind to the biotin-C35.Virus constructs prepared as described above were added to the beads.The beads were collected using a magnet, and the unbound virus wascollected separately. The beads were washed 5× with 1 ml PBS. All of thewashes with the unbound virus were pooled (“Unbound”). The beads wereremoved from the magnet. 1 ml of DMEM supplemented with 2.5% FBS wasadded and the solution was transferred to a fresh tube (“Bound”).“Unbound” and “Bound” were titered. The results are shown in Table 1.These results show that EEV expressing the 2408 antibody, C35-specific,bound to the beads while the EEV expressing the 7000 antibody,VEGF-specific, did not bind.

TABLE 1 Selection of C35-specific mAb using Biotin-C35 and SAV magneticbeads Virus Titer % Bound MAb 7000 Unbound 1.45 × 10⁷  MAb 7000 Bound1.2 × 10⁴ 0.1%  MAb 2408 Unbound 7.6 × 10⁶ MAb 2408 Bound 7.7 × 10⁶ 50%

Spiking experiments were performed where EEV expressing L517 was set atmoi=1, and co-infected into Hela with a mix where EEV expressingH2124-A56R was diluted to 1:10{circumflex over ( )}4 and 1:10{circumflexover ( )}5 with H7000-A56R (one T175 Hela per spiking condition). Inshort, EEV was harvested, and 500 μl EEV containing supernatant(5×10{circumflex over ( )}6 pfu) was used for each spike. 500 μl DMEMcontaining 1 μg Biotin-hC35 was added to each sample (1 ml volume @ 1ug/ml concentration). The Bound and Unbound solutions were collectedusing the SAV-bead (M280) selection method described above. Bound viruswas amplified on BSC1 in T75 flasks.

The collected Bound and Unbound samples for each spiking experiment weretested for enrichment by flow cytometry. The results from the spikingexperiments showed a clear enrichment with beads 10⁻⁴ and 10⁻⁵ and thatbead selection was more efficient than the plate selection method (datanot shown).

Different beads were also tested, Protein G beads (Dynal) andtosylactivated beads (Dynal) using methods similar to those describedabove for SAV beads. The following previously identified antibodies wereused during the selection assays: MAb 2408 (C35-specific antibody, ahumanized 1F2 antibody comprising H2124+L517), MAb 2368 (CD100-specificantibody, disclosed in U.S. Appl. No. 2010/0285036), mAb 7000(VEGF-specific parent antibody of bevacizumab), and mAb 8000(Her2-specific parent antibody of trastuzumab).

Protein G Bead Selection.

EEV produced in small scale infections of Hela cells in 6 well plates(titer ˜5×10{circumflex over ( )}5/ml) were used. Protein G beadselection was tested using EEV expressing 2368-A56R (H2090-A56R+L512,both VH and VL expressed in vaccinia): 1 ml virus (˜5×10{circumflex over( )}5 pfu) and EEV expressing 2408-A56R (H2124-A56R+L517, both VH and VLexpressed in vaccinia): 1 ml virus (5×10{circumflex over ( )}5 pfu)).CD100 bound to Protein G beads was prepared as follows: 300 μl magneticProtein G beads (2× standard amount/sample) were used and pull down wasperformed with a magnet. 600 μl PBS+18 μl CD100-Fc (=36 μg) was added tothe beads, which were incubated at room temp for 20 minutes (on rotator)to allow CD100-Fc to bind to Protein G beads. Beads were pulled downwith a magnet and washed 1× with 1 ml PBS. Next, the beads wereresuspended in 300 μl DMEM supplemented with 10%. 100 μl CD100-Fc/Pro Gbeads were added to each virus sample (˜2× the standard amount of Pro-Gbeads), which was about 12 μg/ml CD100-Fc. The solution was incubatedfor 2 hours at room temperature. 550 μl (about 50%) of the beads wereremoved and unbound was collected following standard 5×1 ml PBS washes.Beads were removed from the magnet, 1 ml DMEM supplemented with 2.5% wasadded, and the solution was transferred to a fresh tube (“Bound”).“Unbound” and “Bound” were titered. The remaining 550 μl was allowed tocontinue incubating at room temp for another 1.5 hours (3.5 hours total)and then for 18 hours at 4 degrees before being harvested as describedabove.

Tosylactivated Bead Selection.

EEV expressing the same 2408 (C35-specific) and 2368 (CD100-specific)antibodies used in the Protein G bead selection experiments above wereused for the tosylactivated magnetic bead selection. 100 μg C35-His wasconjugated to tosylactivated magnetic beads in PBS or ELISA coatingbuffer (CB). The solution was incubated at 37 degrees overnight, andblocked for 1 hour at 37 with PBS, 10% FBS, 0.5% BSA. The beads werewashed 1×, resuspended in 160 μl DMEM supplemented with 10%. 50 μl ofeach bead sample was added to each virus sample and incubated at roomtemp for 5 hours. Unbound was collected following standard 5×1 ml PBSwashes. Beads were removed from the magnet, 1 ml DMEM supplemented with2.5% was added, and the beads were transferred to fresh tube (“Bound”).“Unbound” and “Bound” were titered.

100 μg CD100-His was conjugated to tosylactivated magnetic beads in PBSfor the CD100 antibody selection assay with 2368-A56R (1 ml virus(5×10{circumflex over ( )}5 pfu)) and 2408-A56R (1 ml virus(5×10{circumflex over ( )}5 pfu)) using the same methods described abovefor the C35 antibody selection assay.

The results using the Protein G bead selection are shown in in Table 2and the results using tosylactivated bead selection are shown in Tables3 and 4.

TABLE 2 Selection of CD100-specific mAb using CD100-Fc and Protein Gbeads Virus/Binding time Sample Titer % Bound MAb 2408 - 2 hours Unbound100,000 MAb 2408 - 2 hours Bound 360 0.36% MAb 2368 - 2 hours Unbound64,000 MAb 2368 - 2 hours Bound 88,000   58% MAb 2368 - overnightUnbound 130,000 MAb 2368 - overnight Bound 90,000   41% MAb 2408 -overnight Unbound 320,000 MAb 2408 - overnight Bound 160 0.05%

TABLE 3 Selection of C35-specific mAb using C35 Tosylactivated beadsVirus Sample Titer % Bound MAb 2408 Unbound 96,000 MAb 2408 Bound160,000 61% MAb 2368 Unbound 240,000 MAb 2368 Bound 1,600 0.6%  MAb 2408Unbound 97,000 MAb 2408 Bound 140,000 59%

TABLE 4 Selection of CD100-specific mAb using CD100-His Tosylactivatedbeads Virus Sample Titer % Bound MAb 2408 Unbound 384,000 MAb 2408 Bound480  0.1% MAb 2368 Unbound 264,000 MAb 2368 Bound 232,000 46.7%

Example 4: CH1-A56R Fusion Protein Library Creation

A library of polynucleotides encoding immunoglobulin segments wasproduced as follows. A recombinant vaccinia library referred to as“naïve heavy, A56R fusion” was created using bone marrow RNA that waspurchased from a commercial supplier (Life Technologies) representingmore than 100 donors. Reverse transcription was performed usingantisense primers specific for the constant region of either humanimmunoglobulin gamma or mu. The resulting cDNA was used as template forPCR with one of two sense primers that bound to the beginning of humanvariable heavy framework region 1 and introduced a BssHII restrictionsite in combination with a pool of antisense primers that bound to thevarious germline human J segments and introduced a BstEII restrictionsite. The sequences of these primers were as follows:

Sense VH 3: (SEQ ID NO: 12) AATATGCGCGCACTCCGAGGTGCAGCTGGTGGAGTCTGGSense VH 3a: (SEQ ID NO: 13) AATATGCGCGCACTCCGAGGTGCAGCTGTTGGAGTCTGGAntisense JH 1: (SEQ ID NO: 14) GAGACGGTGACCAGGGTGCCCTGGCCCCA AntisenseJH 2: (SEQ ID NO: 15) GAGACGGTGACCAGGGTGCCACGGCCCCA Antisense JH 3: (SEQID NO: 16) GAGACGGTGACCATTGTCCCTTGGCCCCA Antisense JH 4/5: (SEQ ID NO:17) GAGACGGTGACCAGGGTTCCCTGGCCCCA Antisense JH 6: (SEQ ID NO: 18)GAGACGGTGACCGTGGTCCCTTGGCCCCA

The resulting PCR products were cloned into the pJEM1 plasmid disclosedabove for the purpose of creating recombinant vaccinia virus. Inparticular, the human immunoglobulin variable heavy expression cassettedescribed herein was cloned in frame with human immunoglobulin constantdomain region CH1 and vaccinia virus integral membrane protein A56RcDNA. The resulting proteins created from expression of the library werefusion proteins containing an immunoglobulin heavy chain variablesegment, the heavy chain CH1, and a portion of the A56R proteinexpressed on the surface of vaccinia EEV.

The naïve heavy, A56R fusion library was used along with vacciniaexpressing known Ig-L or a vaccinia virus expressed Ig-L library (aspreviously disclosed in U.S. Pat. No. 7,858,559, which is incorporatedherein by reference in its entirety) for vaccinia panning as illustratedin FIG. 2.

Example 5: CH1-A56R Fusion Protein Library Screening for CD100 AntibodySelection

Selection for new CD100 antibodies using the ˜1,200,000 clones from thenaïve heavy, A56R fusion library (also referred to as “library 3”)described in the previous Example+light chain clones (L48, L116 andL9021) was performed.

T-175 Hela cells were infected with EEV expressing the fusionlibrary+Light chains described above for 2 days after which thesupernatant was harvested, pelleted with low speed spins 2×, and the EEVpelleted at 15,000 RPM for 1 hour. EEV was resuspended in 3 ml DMEMsupplemented with 10% FBS.

Round 1 Selection. EEV expressing 2368-A56R (1 ml virus(˜5×10{circumflex over ( )}5 pfu)) and EEV expressing 2408-A56R (1 mlvirus (5×10{circumflex over ( )}5 pfu)) were used as controls andlibrary 3 (1 ml virus (˜10{circumflex over ( )}8 pfu)) was used for theselection assay. First, 300 μl Protein G beads (2× standardamount/sample) were pulled down with a magnet, and 600 μl PBS+18 μlCD100-Fc (=36 μg) was added to the beads. The solution was incubated atroom temp for 20 minutes (on rotator) to allow CD100-Fc to bind toProtein G beads. Beads were pulled down with magnet, washed 1× with 1 mlPBS, and resuspended in 300 μl DMEM supplemented with 10%.

Next, 100 μl of the CD100-Fc/Pro G per sample (about 12 μg/ml CD100-Fc)was added to the EEV (2408 and 2368 controls, and library 3) andincubated for 2 hours at room temperature. 550 μl (about 50%) of thebeads were removed and unbound virus was collected following standard5×1 ml PBS washes. Beads were removed from the magnet and 1 ml DMEMsupplemented with 2.5% was added, and the solution was transferred to afresh tube (“Bound”). “Unbound” and “Bound” were titered. These “2 hourincubation” samples were titered with methyl cellulose added after 45minutes. Beads recovered from the bound library were amplified on BSC1in T75 (This Round 1 2 hour selection was termed “CD100 3.1A”). Theother 550 μl (about 50%) of the beads was allowed to continue incubatingat room temp for another 1.5 hours (3.5 hours total) and then for 18hours at 4° C. degrees (“overnight”). The unbound virus was collectedfollowing standard 5×1 ml PBS washes. Beads were removed from themagnet, 1 ml DMEM supplemented with 2.5% was added and the solution wastransferred to a fresh tube (“Bound”). “Unbound” and “Bound” weretitered. Bound library was amplified on BSC1 in T75 (This Round 1overnight selection was termed “CD100 3.1B”). The results are shown inTable 5.

TABLE 5 Round 1 Selection of CD100 Ab % Virus/Binding time Sample TiterBound 2408-2 hours Unbound 100,000 2408-2 hours Bound 360 0.36% 2368-2hours Unbound 64,000 2368-2 hours Bound 88,000  58% Library 3.1A-2 hoursUnbound 22,000,000 Library 3.1A-2 hours Bound 20,000 ~0.1%2408-Overnight Unbound 130,000 2408-Overnight Bound 90,000  41%2368-Overnight Unbound 320,000 2368-Overnight Bound 160 0.05% LibraryUnbound 56,000,000 3.1B-Overnight Library Bound 17,000 0.03%3.1B-Overnight

Library 3.1A and 3.1B gave good amplification on BSC1, harvest and titer(˜2×10{circumflex over ( )}7/ml each).

Round 2 Selection. EEV produced in small scale infections of Hela in 6well plates (titer ˜5×10{circumflex over ( )}5/ml) were used. Library3.1A and 3.1B were pooled together into one sample. EEV expressing2368-A56R (1 ml virus (˜5×10{circumflex over ( )}5 pfu)), EEV expressing2408-A56R (1 ml virus (5×10{circumflex over ( )}5 pfu)) and 3.1A/Blibrary (1 ml virus (˜5×10{circumflex over ( )}5 pfu)) were eachcombined with 300 μl Protein G beads (2× standard amount/sample). 600 μlPBS+18 μl CD100-Fc (=36 μg) was added to the beads. The solution wasincubated at room temp for 20 minutes (on rotator) to allow CD100-Fc tobind to Protein G beads. The beads were washed and resuspended asdescribed above for Round 1. 100 μl CD100-Fc/Pro G per sample (˜12 μg/mlCD100-Fc) was added to the virus samples and incubated for 4.5 hours atroom temperature. The “Unbound” and “Bound” were collected and titered.Bound library was amplified on BSC1 in T75 (Round 2 selection was termed“CD100 3.2”). The results of the Round 2 selection are shown in Table 6.

TABLE 6 Round 2 Selection for CD100 Ab Virus-Antigen Sample Titer %Bound 2408-CD100-Fc Unbound 384,000 2408- CD100-Fc Bound 780 0.2% 2368-CD100-Fc Unbound 264,000 2368- CD100-Fc Bound 224,000  46% LibraryUnbound 780,000 3.2-CD100-Fc Library Bound 5,000 0.6% 3.2-CD100-Fc

Library 3.2 gave good amplification on BSC1, harvest and titer(˜3×10{circumflex over ( )}7/ml), and resulted in a small population ofpositive cells. A third round of selection was performed.

Round 3 Selection. A third round of selection was performed using thesame methods described above using “library 3.2A” (Rounds 1 and2=CD100-Fc/Pro G). Bound library was amplified on BSC1 in T75 (Round 3selection was termed “CD100 3.3A”). The results of the Round 3Aselection were tested by flow cytometry. A second Round 3 selection wasperformed with 100 μg CD100-His conjugated to tosylactivated magneticbeads in PBS using the methods disclosed above. 50 μl per sample wasadded for selection using the same lot of virus that was used for CD1003.3A (2368-A56R (1 ml virus (˜5×10{circumflex over ( )}5 pfu)),2408-A56R (1 ml virus (5×10{circumflex over ( )}5 pfu)) and 3.2A (1 mlvirus (˜5×10{circumflex over ( )}5 pfu)). The solutions were incubatedat room temperature for 4 hours. The “Unbound” and “Bound” werecollected and titered. Bound library was amplified on BSC1 in T75 (Round3 tosylactivated selection was termed “CD100 3.3B”). The results of theround 3B selection were tested by flow cytometry. A diagram summarizingthe CD100 antibody selection strategy is illustrated in FIG. 6.

Flow cytometry staining suggested that there was probably a positivepopulation in CD100 3.3A/B when paired with L116. Plaques from 3.3A(n=27) and 3.3B (n=30) were picked and amplified for 3 days on BSC1 in24-well plate (1 plaque per well). Hela cells were infected in 24-wellplates with ⅓ of each amplified plaque. The cells were co-infected withL116 at moi=1 (controls: 2368, 2408 and uninfected Hela supernatant).EEV was produced for 2 days, harvested, and inactivated with psoralenand irradiation with long-wave UV light (PLWUV). The virus was bound toCD100 (2 μg/ml) and C35 (2 μg/ml) coated plates O/N using 50 μl EEV+50μL ELISA blocking buffer per well.

Antibody binding was detected by adding anti-Fab-HRP. Two clones(3.3.C20 and 3.3C27) had good binding to CD100 and were sequenced. Theseclones were further characterized by flow cytometry for specificity andaffinity. The clones were amplified on BSC1 in T75, and titered. The3.3A/B (with L116) infected cells were CD100 sorted. The virus fromsorted cells (150 cells) was amplified, titered and tested by flow (100μg/ml CD100-His: 30 minutes on ice, washed with 5 ml, followed byanti-HIS-APC+anti-Fab-FITC: 30 minutes on ice). Both Clones 20 and 27bound to CD100 as determined by flow cytometry.

The sequences for two high affinity CD100 VH clones (3.3.C20 and 3.3C27)when paired with L116 were identical. The sequence alignment of the twoclones is shown in FIG. 7. The amino acid sequence for the variableheavy chain is as follows (VH CDR1-3 are underlined):

(SEQ ID NO: 19) EVQLVESGGGLVKPGGSLRLSCAASGFIFTDYYLSWIRQAPGKGPEWLSYISSYSRYTNYADSVKGRFTISRDNTRNSIYLQMNNLRVEDTAVYYCARAG SYYGYWGQGTLVT.

Example 6: CH1-A56R Fusion Protein Library Screening for Her2 AntibodySelection

Selection for new Her2 antibodies using 1,200,000 clones from the naïveheavy, A56R fusion library (also referred to as “library 3”)+light chainclones (L48, L116 and L9021) was performed. The library is the same thatwas used for the CD100 selections discussed above.

Round 1 Selection. Library 3 (1 ml virus (˜10{circumflex over ( )}8pfu)) was used for this selection. First, 100 μl PBS+100 μl Her2-Fc (R&DSystems) (=10 μg) was added to the Protein G beads. The solution wasincubated at room temp for 25 minutes (on rotator) to allow Her2-Fc tobind to Protein G beads. Beads were pulled down with magnet, washed 1×with 1 ml PBS, and resuspended in 100 μl DMEM supplemented with 10%.

Next, 100 μl Her2-Fc/Pro G (˜10 μg/ml Her2-Fc) was added to 1 ml ofLibrary 3 and incubated for 4 hours at room temperature. Beads wereremoved and unbound virus was collected following standard 5×1 ml PBSwashes. Beads were removed from the magnet and 1 ml DMEM supplementedwith 2.5% was added, and the solution was transferred to fresh tube(“Bound”). “Unbound” and “Bound” were titered. Beads recovered from thebound library were amplified on BSC1 in T75 (Round 1 selection wastermed “Her2.3.1”).

Round 2 Selection. Amplified Her2.3.1 was titered and amplified in 6well plate format (co-infected with L48, L116 and L9021) and anadditional cycle of Her2-Fc/ProG selection was performed using themethods described above. Bound library was amplified on BSC1 in T75(Round 2 selection was termed “Her2.3.2”).

Round 3 Selection. Amplified Her2.3.2 was titered and reamplified in 6well plate format (co-infected with L48, L116 and L9021) and anadditional cycle of Her2-Fc/ProG selection was performed using themethods described above. Bound library was amplified on BSC1 in T75(Round 3 selection was termed “Her2.3.3”). The results of the Her2.3.2and Her2.3.3 selection were tested by flow cytometry. In thisexperiment, 3 μg/ml C35-His or 10 μg/ml Her2-His were incubated withanti-His-APC MAB for 30 minutes on ice to form complexes. Anti-Fab-FITCas then added and the Antigen-anti-His complexes were added to the cellsfor 30 minutes on ice. The cells were then washed with 2 ml PBS, 0.5%BSA, 2 nM EDTA. Anti-his-APC and anti-Fab-FITC were then added for 30minutes on ice, the cells were then washed, fixed, and flow cytometryassay was run. As shown in FIG. 8, all three light chains enriched forHer2 specific antibodies.

Round 4 Selection. Hela cells in 6 well plate format were co-infectedwith Her2.3.3 and L116 only, EEV was isolated as described above and anadditional cycle of Her2-Fc/ProG selection was performed using themethods described above. Bound library was amplified on BSC1 in T75(Round 4 selection was termed “Her2.3.4”). A diagram summarizing theHer2 antibody selection strategy is illustrated in FIG. 9.

The results of the Her2.3.3 and Her2.3.4 selection were tested by flowcytometry using the staining method described above. ControlH8000-A56R+L8000 was used (8000=chimeric 4D5; the mouse parent oftrastuzumab).

The flow cytometry results showed 2 populations in the 3.3 and 3.4samples. The Her2 3.4 sample was co-infected into Hela cells and thesample stained for Her2 binding and positive cells sorted. Clones werepicked from the sorted sample and screened 30 plaques were picked fromHer2.3.4/sort and amplified for 2 days on BSC1 in 24-well plate (1plaque per well). Hela cells were infected in 24-well plates with ⅓ ofeach amplified plaque The cells were co-infected with L116 at moi=1(controls: 8000, 2368, 2408 and uninfected Hela supernatant). EEV wasproduced for 3 days, harvested, and inactivated with PLWUV. The viruswas bound to CD100 (2 μg/ml) and Her2 (2 μg/ml) coated plates O/N using50 μL EEV+50 μL ELISA blocking buffer per well. The results are shown inFIG. 10.

Antibody binding was detected by adding anti-Fab-HRP. Five positiveclones were identified with good binding to Her2 and were sequenced. All5 clones had the same sequence (see FIG. 11). The VH sequence of cloneB10 is shown below.

Her2 B10 clone Sequence:

(SEQ ID NO: 20) EVQLLESGGGFVQPGGSLRLSCAASGFAFNNYALSWVRQAPGRGLKWVSAISPDGDYIYYADSVKGRFIFSRDNSRNMLSLQMTSLGAEDTALYYCARQNNVRDGAVAGPLDHWGQGTLVT.

Example 7: CH1-A56R Fusion Protein Library Screening for C35 AntibodySelection

Selection for new C35 antibodies using ˜1,200,000 clones from the naïveheavy, A56R fusion library (also referred to as “library 3”)+light chainclones (L48, L116 and L9021) was performed. The library is the same thatwas used for the CD100 and Her2 selections discussed above.

Round 1 Selection.

100 μg C35 was conjugated to tosylactivated magnetic beads in PBS orELISA coating buffer (CB). The solution was incubated at 37° C.overnight, and blocked for 1 hour at 37° C. with PBS, 10% FBS, 0.5% BSA.The beads were washed 1×, resuspended in 160 μl DMEM supplemented with10%. 50 μl of each bead sample was added to each virus sample andincubated at room temp for 3.5 hours. Unbound was collected followingstandard 5×1 ml PBS washes. Beads were removed from the magnet, 1 mlDMEM supplemented with 2.5% was added, and the beads were transferred tofresh tube (“Bound”). “Unbound” and “Bound” were titered.

Bound library was amplified on Hela in T75 (Round 1 selection was termed“C35 3.1”). The results of the round C35 3.1 were tested by flowcytometry. C35 3.1 bound, but was low (data not shown).

Round 2 Selection. Amplified C35 3.1 was titered and used to producerecombinant EEV in 6 well plate format by co-infection of C35 3.1 withL48, L116 and L9021 (titer ˜5×10{circumflex over ( )}5/ml) and anadditional cycle of tosylactivated C35 selection was performed using themethods described above. Solutions were incubated at room temp for 3.0hours instead of 3.5 as in Round 1. The titers of bound and unboundvirus are shown in Table 7. Bound library was amplified on Hela in T75(Round 2 selection was termed “C35 3.2”) and binding was tested by flowcytometry as described above.

TABLE 7 Round 2 C35-His/Tosylactivated Selection for C35 Ab Virus SampleTiter % Bound 2368 Unbound 684,000 2368 Bound 1600 0.2%  2408 Unbound600,000 2408 Bound 168,000 28% Library C35 3.2 Unbound 972,000 LibraryC35 3.2 Bound 10,400  1%

Round 3 Selection. Amplified C35 3.2 was titered and used to producerecombinant EEV in 6 well plate format by co-infecting with L48, L116and L9021 (titer ˜5×10{circumflex over ( )}5/ml) and an additional cycleof tosylactivated C35 selection was performed using the methodsdescribed above for Round 2. The titers of bound and unbound virus areshown in Table 8. Bound library was amplified on Hela in T75 and testedfor C35 binding by flow cytometry as above (Round 3 selection was termed“C35 3.3”).

TABLE 8 Round 3 C35-His/Tosylactivated Selection for C35 Ab Virus SampleTiter % Bound 2368 Unbound 400,000 2368 Bound 480 0.1% 2408 Unbound228,000 2408 Bound 108,000  47% Library C35 3.3 Unbound 540,000 LibraryC35 3.3 Bound 2600 0.5%

Clones will be screened from C35 3.3 as well a possible fourth roundselection. Positive clones will be characterized by flow cytometry andtested for specificity, affinity, and function.

Example 8: Selective Amplification of Vaccinia Virus Expressing Heavy orLight Chains

Combinatorial infection with separate recombinant vaccinia virusesharboring either heavy or light chain immunoglobulin is an effective wayto express antibodies for selection. However, post-selection, duringamplification and harvest, there is currently no mechanism forseparating heavy and light chain-containing viruses. Therefore, it wouldbe advantageous to be able to amplify heavy and light-containingvaccinia viruses separately as in the instance where both heavy andlight chain infections are conducted at complexities of greater than oneand where deconvolution post-selection is required. For this reason,recombinant vaccinia viruses expressing either heavy or light chaincoupled to a drug selectable marker (heavy chain with neomycinresistance and light chain with hygromycin resistance) have beenproduced. The following experiment demonstrates utility in selectivelyamplifying heavy or light chain-containing recombinant vaccinia virusesindependently.

BSC1 cells were seeded out into 15 wells of 6-well plates at 1.25×10⁶cells per well and at 2.5 ml per well. The next day, a series ofdilutions of hygromycin or G418 for selection was created according toTable 9. DMEM-2.5 represents DMEM containing 2.5% FBS.

TABLE 9A Preparation of hygromycin dilutions Hygromycin Dilutions[stock] = 50 mg/ml 1 2 3 4 5 6 0.2 0.1 0.08 0.04 0.02 0.01 mg/ml mg/mlmg/ml mg/ml mg/ml mg/ml Culture vol. (ml) 6 6 12 0.5 X 0.5 X 0.5 Xneeded: serial serial serial Add Hygro (μl): 24 12 19.2 6 ml of 3 6 mlof 4 6 ml of 5 To DMEM-2.5 (ml): 5.976 5.988 5.9808 into 6 ml into 6 mlinto 6 ml DMEM-2.5 DMEM-2.5 DMEM-2.5

TABLE 9B Preparation of G418 dilutions G418 Dilutions [stock] = 100mg/ml 1 2 3 4 5 6 2.0 1.0 0.5 0.25 0.125 0 mg/ml mg/ml mg/ml mg/ml mg/mlmg/ml Culture vol. (ml) 5 5 5 5 5 5 needed: Add G418 (μl): 200 5 ml of 15 ml of 2 5 ml of 3 5 ml of 4 5 ml of To DMEM-2.5 (ml): 10 into 5 mlinto 5 ml into 5 ml into 5 ml DMEM-2.5 DMEM-2.5 DMEM-2.5 DMEM-2.5DMEM-2.5

On the third day, the BSC1 cells were infected with MOI=3 of eitherwild-type vaccinia virus or vaccinia virus containing the respectiveselectable markers (VHE H5 LX-IRES-HYGRO or VHE H5 HX-A56R NEO).Hygromycin and G418 dilutions were then applied to the plate wells atthe same time. DMEM-2.5 containing no antibiotics was added to thecontrol wells. The infection was carried out in a volume of 0.65 ml perwell and the cells were incubated at 37° C. After 2 hours, the mediavolumes were brought up to 2.65 ml per well and additional hygromycin orG418 was supplemented to maintain intended concentrations in thedrug-containing wells. Meanwhile, new BSC1 cells were seeded into12-well plates at 2×10⁵ cells per well for post-infection titerdetermination.

24 hours post infection, all samples were harvested into 15 ml conicalcentrifuge tubes, freeze-thawed three times, vortexed, and resuspendedby gentle vortexing into 1.8 ml DMEM-2.5. Samples were sonicated for 2minutes at the maximum intensity and then transferred to a 2.0 mlSarstedt tube. A series of dilutions was prepared for each sample in 7.5ml polypropylene tubes. First, 30 μl of the original was withdrawn fromeach sample and combined with antibiotics-free DMEM-2.5 to a finalvolume of 3000 μl (1:10² dilution). Next, 30 μl of the 1:10² dilutionwas added to a second final volume of 3000 μl to prepare a 1:10⁴dilution. A series of 1:10 dilutions was then carried out to prepare the1:10⁵ to 1:10⁹ dilutions. All the dilutions were vortexed in a biosafetycabinet using 5 ml tubes.

The BSC1 cells in the titer plates were subsequently infected using sixdilutions (1:10⁴ to 1:10⁹) from each sample by dispensing 0.333 ml ofeach titer dilution per assay well in duplicates. Therefore, the factorto calculate titer is equal to the total plaque number in 2 duplicatewells divided by 0.66 ml. The infection was incubated for at least 2hours at 37° C. An additional 1.0 ml of DMEM-2.5 was added to each wellafter the initial 2 hours of adsorption and infection.

48 hours post infection, Crystal Violet was added to the 12-well titerplates. Only plaques greater than 1 mm diameter were counted. Daughterplaques were excluded from counting.

The results are shown in Table 10. In hygromycin resistance experiments,0.01 to 0.08 mg/ml of hygromycin significantly inhibited theamplification of vaccinia virus expressing heavy chain linked to aneomycin resistance marker, but had little or no inhibition effect(except for the 0.04 mg/ml data point) on the amplification of vacciniavirus expressing light chain linked to a hygromycin resistance markeruntil the hygromycin concentration was increased to 0.1 to 0.2 mg/ml.Similarly, in neomycin resistance experiments, 0.125 to 2 mg/ml of G418significantly inhibited the amplification of wild-type vaccinia virus,but had no inhibition effect on the amplification of vaccinia virusexpressing heavy chain linked to a neomycin resistance marker.

TABLE 10A Results of hygromycin resistance experiments HYGRO RESISTANCESample ID Titer % Inhibition Hygro 0.2 mg/ml VKE H5 LX-IRES-HYGRO2.20E+07 53.0 Hygro 0.1 mg/ml VKE H5 LX-IRES-HYGRO 1.70E+07 63.6 Hygro0.08 mg/ml VKE H5 LX-IRES-HYGRO 4.47E+07 4.5 Hygro 0.04 mg/ml VKE H5LX-IRES-HYGRO 2.77E+07 40.9 Hygro 0.02 mg/ml VKE H5 LX-IRES-HYGRO4.66E+07 0.4 Hygro 0.01 mg/ml VKE H5 LX-IRES-HYGRO 5.98E+07 −27.9 Hygro0.08 mg/ml VHE H5 HX-A56R NEO 2.43E+06 89.1 Hygro 0.04 mg/ml VHE H5HX-A56R NEO 2.66E+06 88.1 Hygro 0.02 mg/ml VHE H5 HX-A56R NEO 2.70E+0687.9 Hygro 0.01 mg/ml VHE H5 HX-A56R NEO 6.86E+06 69.3 [no antibiotic]VKE H5 LX-IRES-HYGRO 3.86E+07 Hygro control #1 [no antibiotic] VKE H5LX-IRES-HYGRO 5.49E+07 Hygro control #2 [no antibiotic] VHE H5 HX-A56RNEO 2.23E+07 Neo control

TABLE 10B Results of neomycin resistance experiments NEO RESISTANCESample ID Titer % Inhibition G418 0.125 mg/ml WT 1.58E+07 60.8 G418 0.25mg/ml WT 8.26E+06 79.4 G418 0.5 mg/ml WT 2.54E+06 93.7 G418 1 mg/ml WT1.36E+06 96.6 G418 2 mg/ml WT 1.59E+05 99.6 G418 0.125 mg/mlVHE H5HX-A56R NEO 2.88E+07 −26.7 G418 0.25 mg/ml VHE H5 HX-A56R NEO 3.11E+07−36.7 G418 0.5 mg/ml VHE H5 HX-A56R NEO 3.41E+07 −50.0 G418 1 mg/ml VHEH5 HX-A56R NEO 3.18E+07 −40.0 G418 2 mg/ml VHE H5 HX-A56R NEO 3.03E+07−33.3 G418 0 mg/ml WT 4.02E+07 Neo control #1 G418 0 mg/ml VHE H5HX-A56R NEO 2.27E+07 Neo control #2

Therefore, recombinant vaccinia viruses expressing immunoglobulin anddrug resistance markers linked via an Internal Ribosome Entry Site(IRES) provide for protection against death of the host cells undertreatment with that drug. This allows for chain-specific propagation invirus as well as selection against wild-type vaccinia virus duringrecombination.

Example 9: Expression of A56R Fusion Protein on Surface of Hela Cells

HeLa cells were co-infected with recombinant EEV vaccinia virusexpressing immunoglobulin fusion constructs, Variable Heavy (H8000)CH1-A56R with L8000 Ig-K which together encode an Fab fragment ofantibody (“Fab”), Variable Heavy (H8000) FL-A56R with L8000 Ig-K whichtogether encode full length (“FL”) IgG, Variable Heavy (H8000)FL-truncated-A56R with L8000 Ig-K which together encode full length IgGwith a shorter A56R (“TR”), Variable Heavy (H2124) FL-A56R with L517Ig-K (2408 “FL”), and Variable Heavy (H2124) FL-truncated-A56R with L517Ig-K (2408 “TR”) in 12-well plates. A diagram showing the “Fab”, “TR”and “IgG” constructs is shown in FIG. 12. Fluorescence Activated CellSorting (FACS) analysis for C35 staining and Her2 staining of cellsinfected with recombinant vaccinia virus was performed. After ˜18 hourscells were stained with C35-His/His-APC and Her2-His/His-APC, andanti-Fab-FITC; and detected by FLOW analysis on Canto. Briefly, cellswere trypsinized and divided into two per samples; washed with 2 mL washbuffer; 10 μg/ml Her2-His or 4 μg/ml C35-His were added to one of thetwo samples and incubated for 30 minutes on ice. The cells were thenwashed and anti-His APC was added and the samples were incubated for 30minutes on ice, stained with secondary detection reagent anti-Fab-FITC,and then the samples were washed, fixed (0.5% Paraformaldehyde with1:100PI for 20 min on ice) and analyzed by FLOW by Canto. The FACS datais shown in FIG. 13-15. These results show that the A56R fusionproteins, either expressing an Fab or Full Length IgG were expressed onthe cell surface, and that only the transmembrane and intracellulardomains of A56R are necessary for surface expression of IgG.

Example 10: Solution Based Vac-Ig Selection

Tosylactivated bead selection. EEV expressing the C35-specific (H2124)“Fab”, “FL” and “TR” VH were co-infected along with L517 into Hela cellsin 6 well plates, and EEV harvested from supernatant by spinning 1200rpm and collecting supernatant (EEV) after approximately 48 hours ofinfection. As a control Her2 specific H8000-Fab+L8000 was produced thesame way. For bead selection, 100 μg C35-His was conjugated totosylactivated magnetic beads in PBS. The solution was incubated at 37°C. overnight, and blocked for 1 hour at 37° C. with PBS, 10% FBS, 0.5%BSA. The beads were washed 1×, resuspended in 160 μl DMEM supplementedwith 10%. 50 μl of each bead sample was added to each virus sample andincubated at room temperature for 2 hours. Unbound EEV was collected instandard 5×1 ml PBS washes. Beads were removed from the magnet, 1 mlDMEM supplemented with 2.5% was added, and the beads were transferred tofresh tube (“Bound”). “Unbound” and “Bound” were titered.

As shown in Table 11, Vaccinia virus expressing C35-specific constructsof both the Fab and FL fusion proteins were selected, while theconstruct with the TR fusion protein was not. This data suggests thatsome extracellular A56R sequence is needed for incorporation into EEV.

TABLE 11 Results of Tosylactivated bead selection Virus % Bound MAb2408-Fab  24% Mab 2408-FL  18% Mab 2408-TR 2.3% Mab 8000-Fab 0.8%

Example 11: CH1-A56R Fusion Protein Library Screening for CD100 AntibodySelection

Selection for new CD100 antibodies using a heavy chain library comprisedof ˜7,000,000 clones containing a combination of naïve VH and syntheticVH sequences was produced in the A56R-Fab vector. To produce vacciniaexpressing the library of Ig on the surface of EEV, the A56R fusionlibrary (also referred to as “library 10”) was co-infected into1×10{circumflex over ( )}9 Hela cells along with a cocktail of 9 lightchain clones (Kappa Chains: L48, L116, L122, L7110, and L9021; andLambda Chains: L3-1, L151, L214, and L223). The total moi of heavy chainvirus was 1, and the total moi of light chain virus was 1, with eachlight chain comprising approximately 1/9th of the total light chainvirus added.

Hela-S cells growing in suspension were infected for 2 days, after whichthe supernatant was harvested, pelleted with low speed spins 2×, and theEEV pelleted at 13,000 RPM for 1 hour in an F16/F250 rotor. EEV wereresuspended in 3 ml DMEM supplemented with 10% FBS, and 1 ml was used toselect CD100 specific antibodies.

Round 1 Selection.

EEV expressing 2368-A56R (1 ml virus with approximately ˜5×10{circumflexover ( )}5 pfu)) and EEV expressing 2408-A56R (1 ml virus withapproximately 5×10{circumflex over ( )}5 pfu) were used as controls andlibrary 10 (1 ml virus with approximately ˜10{circumflex over ( )}8 pfu)was used for the selection assay. First, 300 μl Protein G beads (2×standard amount/sample) were pulled down with a magnet, and 600 μlPBS+18 μl CD100-Fc (=36 μg) was added to the beads. The solution wasincubated at room temp for 20 minutes (on a rotator) to allow CD100-Fcto bind to Protein G beads. Beads were pulled down with magnet, washed1× with 1 ml PBS, and resuspended in 300 μl DMEM supplemented with 10%FBS.

Next, 100 μl of the CD100-Fc/Pro G per sample (about 12 μg/ml CD100-Fc)was added to the EEV (2408 and 2368 controls, and library 10) andincubated for 2 hours at room temperature. Unbound virus was removedfollowing standard 5×1 ml PBS washes. Beads were removed from the magnetand 1 ml DMEM supplemented with 2.5% was added, and the solution wastransferred to a fresh tube (“Bound”). “Unbound” and “Bound” weretitered. The results are shown in Table 12. Bound virus was amplified onBSC1 cells in T175 flasks for 3 days.

TABLE 12 Results of Tosylactivated bead selection Titer Titer PercentVirus Selection Unbound Bound Bound Library_10 CD100-Fc 1.5 ×10{circumflex over ( )}8 2.2 × 10{circumflex over ( )}6 0.15 2368CD100-Fc 72,000 37,000 34 2408 CD100-Fc 338,400 80 0.12

These results show that Library 10.1 gave good amplification on BSC1,harvest and titer.

Round 2 Selection.

EEV from 10.1+a fresh aliquot of the 9 Light chains was produced byinfecting Hela cells in a cell stacker at moi=1 each for 2 days, andharvesting as described above. The harvested virus was split in half,with 50% being selected on ProG beads, and 50% being selected on CD100coated Tosyl activated bead.

Round 2 Employing ProG Bead Selection.

EEV expressing 2368-A56R (1 ml virus (˜5×10{circumflex over ( )}5 pfu)),EEV expressing 2408-A56R (1 ml virus (5×10{circumflex over ( )}5 pfu))and 10.1 library (1 ml virus (5×10{circumflex over ( )}5 pfu)) were usedfor selection. 600 μl PBS+18 μl CD100-Fc (=36 μg) was added to the Pro-Gbeads. The solution was incubated at room temperature for 20 minutes (ona rotator) to allow CD100-Fc to bind to Protein G beads. The beads werewashed and resuspended as described above for Round 1. 100 μlCD100-Fc/Pro G per sample (˜12 μg/ml CD100-Fc) was added to the virussamples and incubated for 2 hours at room temperature. The “Unbound” and“Bound” were collected and titered. Bound library was amplified on BSC1in T75 (Round 2 selection was termed “CD100 10.2/ProG”). The results ofthe Round 2 selection are shown in Table 13A. Bound virus was amplifiedon BSC1 in a T175 flask for 3 days.

Round 2 Employing Tosylactivated Bead Selection.

EEV expressing the same 2408 (C35-specific), 2368 (CD100-specific) andLibrary 10 antibodies used in the Protein G bead selection experimentsabove were used for the tosylactivated magnetic bead selection. 100 μgCD100-His was conjugated to tosylactivated magnetic beads in PBS. Thesolution was incubated at 37° C. overnight, and blocked for 1 hour at 37with PBS, 10% FBS, 0.5% BSA. The beads were washed 1× with DMEM, 10%FBS, resuspended in 160 μl DMEM supplemented with 10% FBS. 50 μl of eachbead sample was added to each virus sample and incubated at roomtemperature for 2 hours. Unbound virus was collected in standard 5×1 mlPBS washes. Beads were removed from the magnet, 1 ml DMEM supplementedwith 2.5% was added, and the beads were transferred to a fresh tube(“Bound”). “Unbound” and “Bound” were titered. Bound virus was amplifiedon BSC1 in a T175 flask for 3 days. The results of the Round 2 selectionare shown in Table 13B.

TABLE 13A Round 2 Selection for CD100 Ab (Protein G Bead Selection)Titer Titer Percent Virus Selection Unbound Bound Bound Library_10.1CD100-Fc 4.4 × 10{circumflex over ( )}7 67,000 0.15 Protein G 2368CD100-Fc 104,400 66,000 38.7 2408 CD100-Fc 240,000 80 0.03

TABLE 13B Round 2 Selection for CD100 Ab (Tosylactivated Bead Selection)Titer Titer Percent Virus Selection Unbound Bound Bound Library_10.1CD100-His 2.4 × 10{circumflex over ( )}7 113,000 0.47 Tosyl 2368CD100-His 56,400 106,000 34.4 2408 CD100-His 354,000 0 0

These second round results show that Library 10.2/ProG and 10.2/Tosylboth gave good amplification on BSC1.

Round 3 Selections.

A third round of selection was performed using the same methodsdescribed above. 10.2/ProG was selected with CD100-Fc/ProG for the thirdround, and 10.2/Tosyl was selected with CD100-His/Tosyl for the thirdround. EEV from 10.2/ProG+a fresh aliquot of the 9 Light chains wasproduced by infecting Hela cells in a T175 at moi=1 each for total heavychain and total light chain recombinant virus for 2 days, and harvestingas described above. EEV from 10.2/Tosyl+a fresh aliquot of the 9 Lightchains was produced by infecting Hela cells in a T175 flask at moi=1each for total heavy chain and total light chain recombinant virus for 2days, and harvesting as described above. Titers are shown in Table14A-B.

TABLE 14A Round 3 Selection for CD100 Ab (Protein G Bead Selection)Titer Titer Percent Virus Selection Unbound Bound Bound Library_10.2CD100-Fc 2.5 × 10{circumflex over ( )}7 364,000 1.44 Protein G 2368CD100-Fc 84,000 58,000 48.5 2408 CD100-Fc 99,600 0 0

TABLE 14B Round 3 Selection for CD100 Ab (Tosylactivated Bead Selection)Titer Titer Percent Virus Selection Unbound Bound Bound Library_10.2CD100-His 8.2 × 10{circumflex over ( )}6 6,100 0.074 Tosyl 2368CD100-His 69,600 108,000 60.8 2408 CD100-His 121,000 0 0

Bound library was amplified on BSC1 in T75 (Round 3 selection was termed“CD100 10.3ProG and CD100 10.3/Tosyl”). The results of the Round 3selection were tested by flow cytometry.

In this experiment, an aliquot of the 10.3 selections were co-infectedindividually with each Light chain and then tested for binding to CD100and Her2. Hela cells were infected at moi=1. After an overnightinfection cells were harvested and stained for CD100 binding and Her2binding as control. Cells were trypsinized, washed with ice cold FlowBuffer (FB 1×PBS, 0.5% BSA, 2 mM EDTA) and detected with each of threedifferent detection methods. In the first detection method (2 step)cells were incubated for 30 min with 10 ug/mL huCD100-His in FB on ice,then washed with 2 mL of FB and incubated with 1:50 (2 ug/mL) of Mouseanti 6×His-APC mixed with 1:500 (2 ug/mL) FITC labeled Goat-Fabanti-human-Fab on ice for 30 min. In the second and third detectionmethod (Pre-complexed) either 10 ug/mL of hu CD100-His or 10 ug/mLhuHer2-His were preincubated with 1:50 (2 ug/mL) of Mouse anti 6×His-APCin FB on ice for 30 min, then the mix was added to cells with 1:500 (2ug/mL) GtFab anti huFab-FITC and incubated for 30 min on ice. After theincubation with detection reagents cells were washed 1× with 2 mL FB,reconstituted in 0.5% paraformaldehyde and incubated on ice for 20 min.20,000 events were read on FACS Canto. Results are shown in FIGS. 16-22.

Flow cytometry staining showed that there was a positive population ofCD100 binding cells in CD100 10.3/ProG and Tosyl when paired with mostof the light chains. In particular, a strongly positive population wasobserved when co-infected with L3-1.

In order to isolate the specific VH, Hela cells were separately infectedwith 10.3/ProG or 10.3/tosyl, and co-infected with L3-1. After anovernight infection the cells were harvested and stained for CD100binding with a precomplexed method as described above. Then the antigenbinding cells were isolated by cell sorting. After sorting the virus wasreleased from the cells by freeze/thaw, and then the virus was amplifiedon BSC1 cells. The amplified sample of isolated EEV-VH chains was testedfor enrichment by analytical flow assay. In this assay an aliquot of theamplified sorted CD100 10.3 sample was co-infected with L3-1 Light chainand then tested for binding to CD100 and Her2 with the 2-step andprecomplexed method described above. Results are shown in FIGS. 23-25.

Following amplification the virus was harvested and DNA extracted froman aliquot of the virus using Qiagen DNA blood mini kit (cat #51104).The purified DNA was PCR amplified with Heavy chain specific primers428; 5′-GATATATTAAAGTCGAATAAAGTG-3′ (SEQ ID NO:31) and 430;5′-GACATCACATAGTTTAGTTGC-3′ (SEQ ID NO:32). The resulting PCR productwas cloned into plasmid vector containing secreted full length humanIgG1 (EFVH) and then the V gene contained in the resulting colonies wassequenced. A summary of the sequencing results is shown in Table 15.After sequencing 188 clones from 10.3/ProG, 44 unique clones wereidentified, and after sequencing 188 clones from 10.3/toysl, 46 uniqueclones were identified.

TABLE 15 Summary of Unique Clones Clones unique bound by Screensequenced sequences ELISA 10.3/ProG 188 44 56.8% 10.3/tosyl 188 46 60.9%

Plasmid DNA for each unique heavy chain was co-transfected along with aplasmid vector encoding VL3-1 into CHO cells using Lipofectamine 2000for 3 days, and then the antibody contained in the media was tested forspecificity for CD100 by flow cytometry on CD100+Jurkat cells and byELISA (FIGS. 26 and 27A-B, respectively). For the flow cytometry assay,the experimental antibody was pre-incubated at 1 ug/mL with 1:400 or[2.5 ug/mL] Gt anti Hu Fc-Dylight 649 secondary in Flow Buffer (1×PBS,0.5% BSA, 2 mM EDTA). Jurkat cells were seeded at 250,000/well in 96well plate and incubated with preformed Ab complex for 30 min on ice.The cells were then washed 2× with 200 uL Flow Buffer and incubated for20 min with 0.5% Paraformaldehyde with 1× Propidium Iodide (PI). Cellswere detected on FACS Canto reading 10,000 events gated on live cellpopulation. In total, at least 75 unique antibodies were shown to bespecific for CD100 by ELISA or Flow Cytometry.

Example 12: CH1-A56R Fusion Protein Library Screening for Her2 AntibodySelection

A heavy chain library comprised of ˜3,000,000 clones containing acombination of naïve VH and synthetic VH sequences was produced in theA56R-Fab vector as a fusion with IRES-Neomycin. To produce vacciniaexpressing the library of Ig on the surface of EEV, the A56R fusionlibrary (also referred to as “library 9”) was co-infected along with alibrary of 1,000 Kappa Light chain clones containing a hygromycinresistance gene into 5×10{circumflex over ( )}9 Hela cells. The Lightchain library was comprised of VK sequences isolated from human bonemarrow (naïve). The total moi of heavy chain virus was 1, and the totalmoi of light chain virus was 1.

Hela-S cells growing in suspension were infected for 2 days, after whichthe supernatant was harvested, pelleted with low speed spins 2×, and theEEV pelleted at 13,000 RPM for 1 hour in a F16/F250 rotor. EEV wasresuspended in 3 ml DMEM supplemented with 10% FBS, and 1 ml was used toselect Her2/neu specific antibodies.

Round 1 Selection.

Library 9 was used for this selection. First, 100 μl PBS+24 ug Her2-Fcwas added to 600 ul the Protein G beads. The solution was incubated atroom temperature for 20 minutes (on rotator) to allow Her2-Fc to bind toProtein G beads. Beads were pulled down with magnet, washed 1× with 1 mlPBS, and resuspended in 400 μl DMEM supplemented with 10% FBS.

Next, 100 μl Her2-Fc/Pro G (˜6 μg/ml Her2-Fc) was added to 1 ml ofLibrary 9 and incubated for 2 hours at room temperature. A similaramount of beads were added to positive control MAb 8000 EEV and negativecontrol MAb 2408 EEV. Beads were removed and unbound virus was collectedin standard 5×1 ml PBS washes. Beads were removed from the magnet and 1ml DMEM supplemented with 2.5% FBS was added, and the solution wastransferred to fresh tube (“Bound”). “Unbound” and “Bound” were titered(See Table 16). Beads recovered from the bound library were amplified onBSC1 in three T175 flaks in the presence of 1 mg/ml G418. Thisamplification selected for Heavy chain recombinant virus. (Round 1selection was termed “Her.9.1”).

TABLE 16 Round 1 Selection for Her2 Ab Titer Titer Percent VirusSelection Unbound Bound Bound Library 9 Her2-Fc 1.4 × 10{circumflex over( )}9 4.610{circumflex over ( )}6 0.32 2408 Her2-Fc 1.2 × 10{circumflexover ( )}5 180 0.14 8000 Her2-Fc   3 × 10{circumflex over ( )}5 7.2 ×10{circumflex over ( )}4 19.3

Round 2 Selection.

Amplified Her.9.1 was titered and a second round of selection wasperformed by co-infecting the Her.9.1 VH and a fresh aliquot of theVK1000 Library into Hela cells in 2 CellStackers for 2 days. Virus washarvested as described above and an additional cycle of Her2-Fc/ProGselection was performed using the methods described above. 50% of thebound virus was amplified on BSC1 in two T175 flasks with 1 mg/ml G418(to select for Heavy chains) and 50% of the bound virus was amplified onBSC1 in two T175 flasks with 0.030 mg/ml Hygro (to select for Lightchains). The titer results are shown in Table 17. The amplified viruseswere named Her.9.2/VH and Her 9.2/VK.

TABLE 17 Round 2 Selection for Her2 Ab Titer Titer Percent VirusSelection Unbound Bound Bound Her2 9.Rd1 + VK Her2-Fc 1.76 ×10{circumflex over ( )}8  1.910{circumflex over ( )}5 0.1 2408 Her2-Fc1.4 × 10{circumflex over ( )}5 80 0.1 8000 Her2-Fc 3.3 × 10{circumflexover ( )}5 6.2 × 10{circumflex over ( )}4 16

Round 3 Selection.

Amplified Her.9.2/VH and Her 9.2/VK were titered, co-infected into Helacells in a CellStacker for 2 days, EEV purified as described above, andan additional cycle of Her2-Fc/ProG selection was performed using themethods described above.

50% of the bound virus was amplified on BSC1 in a T175 flask with 1mg/ml G418 (to select for Heavy chains) and 50% of the bound virus wasamplified on BSC1 in a T175 flask with 0.030 mg/ml Hygro (to select forLight chains). The amplified viruses were named Her.9.3/VH and Her9.3/VK.

The selection for Her2 specific antibodies in Her.9.3/VH and Her 9.3/VKwas tested by flow cytometry. Hela cells were co-infected at moi=1 withHer9.3/VH and Her9.3/VK overnight, and then stained for binding to Her2,with the absence of binding to a control antigen (C35). In thisexperiment, 3 μg/ml C35-His or 6 μg/ml Her2-His were incubated withanti-His-APC antibody for 30 minutes on ice to form complexes.Anti-Fab-FITC as then added and the Antigen-anti-His complexes wereadded to the cells for 30 minutes on ice. The cells were then washedwith 2 ml PBS, 0.5% BSA, 2 nM EDTA. The cells were fixed and flowcytometry assay was run. The data showed enrichment of both VH and VK.

To further enrich, fresh Hela cells were infected with the 9.3VH and VKat moi=1 each, the cells were stained as above, and then the antigenbinding cells were sorted. Virus was released from the sorted cells bythree cycles of freeze/thaw and then 50% of the virus was amplified onBSC1 in a T75 flask with 1 mg/ml G418 (to select for Heavy chains) and50% of the virus was amplified on BSC1 in a T75 flask with 0.030 mg/mlHygro (To select for Light chains). The amplified viruses were titeredand named Her.9.3/VH/Sort and Her 9.3/VK/sort.

The selection for Her2 specific antibodies in Her.9.3/VH/sort and Her9.3/VK/sort was tested by flow cytometry. Hela cells were co-infected atmoi=1 with Her9.3/VH/sort and Her9.3/VK/sort overnight, and then stainedfor binding to Her2, with the absence of binding to a control antigen(C35). In this experiment, 3 μg/ml C35-His or 6 μg/ml Her2-His wereincubated with anti-His-APC antibody for 30 minutes on ice to formcomplexes. Anti-Fab-FITC was then added and the Antigen-anti-Hiscomplexes were added to the cells for 30 minutes on ice. The cells werethen washed with 2 ml PBS, 0.5% BSA, 2 nM EDTA. The cells were thenfixed and flow cytometry assay was run. The data in showed enrichment ofboth VH and VK.

In order to fix the antigen specific pairing of VH and VK,Her.9.3/VH/sort and Her 9.3/VK/sort were co-infected into Hela cells atmoi=0.1 each, and then stained as described for binding Her2 above. Thecells were again sorted, but this time individual infected cells weresorted into individual wells of a 96 well plate. Each antigen bindingsorted cell should contain a fixed antigen specific pairing of specificVH with specific VK. After sorting, the cells were subjected tofreeze/thaw, and then the virus was amplified on BSC1 in a 96 wellplate, with virus from one cell being amplified in one recipient well.After 5 days the plates were subjected to freeze/thaw, and then analiquot of virus in each well was infected into Hela cells in 96 wellplate. The virus in each well should contain a mix of VH and VK, and theinfection of Hela cells should result in expression of surface IgG andantigen binding. After an overnight binding the cells were harvested andstained for Her2 binding as described above (FIG. 28).

From screening 1 plate, 26 specific clones were identified. Repeattesting of these clones demonstrated that they bind to Her2, but not C35by flow cytometry. Three representative clones (D5, D8, and H2) are showin FIG. 29A-C. DNA was then extracted from the viruses, and the VH andVK genes contained in these viruses was PCR amplified with VH and VKspecific primers and cloned into mammalian expression vectors so thatthey would be expressed as full length IgG1 and full length Kappa. Thesequences of the VH and VK genes were then determined. By sequencing,these 26 clones contained 15 unique antibodies. These antibodies werethen expressed in CHO cells by co-transfection of the IgG and Kappaexpression plasmids, and antibody was harvested from the cellsupernatant after 3 days. Antibody was quantitated by ELISA, and thentested for specificity by ELISA and flow cytometry on SKBR3 cells(Her2+++). Representative data for antibodies shown to have specificityby ELISA and flow cytometry is shown in FIGS. 30 and 31, respectively.

Repeating the single cell sorting and screening additional clonesaccording to the methods herein resulted in the identification ofadditional novel anti-Her2 antibodies.

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and any constructs, viruses orenzymes which are functionally equivalent are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications are intended to fall within the scope of the appendedclaims.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference. The disclosureand claims of U.S. application Ser. No. 08/935,377, filed Sep. 22, 1997and U.S. Application No. 60/192,586, filed Mar. 28, 2000 are hereinincorporated by reference.

What is claimed is:
 1. A recombinant vaccinia library comprising alibrary of polynucleotides constructed in a vaccinia virus vectorencoding a plurality of immunoglobulin fusion polypeptides, wherein thevaccinia virus vector comprises (a) a first polynucleotide encoding afirst polypeptide segment comprising a heavy chain CH1 domain, (b) asecond polynucleotide encoding a second polypeptide segment comprisingthe the stalk region, the transmembrane domain, and the intracellulardomain of the vaccinia virus EEV-specific A56R protein situateddownstream of the CH1 domain, wherein the second polypeptide segmentcomprises amino acids 215 to 421 of SEQ ID NO: 11 or amino acids 447 to653 of SEQ ID NO: 30, and (c) a third polynucleotide encoding animmunoglobulin heavy chain variable region or fragment thereof situatedupstream of the CH1 domain.
 2. The recombinant vaccinia library of claim1, wherein each immunoglobulin fusion polypeptide further comprises asignal peptide for facilitating expression of the plurality ofimmunoglobulin fusion polypeptides on the surface of EEV.
 3. Therecombinant vaccinia library of claim 1, wherein the first polypeptidesegment further comprises a heavy chain CH2 domain, a heavy chain CH3domain, or a combination thereof.
 4. The recombinant vaccinia library ofclaim 3, wherein the first polypeptide segment comprises a human IgGconstant region, or portion thereof.